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Lars Backman
Protein Chemistry
Also of Interest
Characterization of Biological Membranes.
Nieh, Heberle, Katsaras (Eds.), 
ISBN ----, e-ISBN ----

Bioorganometallic Chemistry.
Weigand, Apfel (Eds.), 
ISBN ----, e-ISBN ----

Methods in Protein Biochemistry.

Tschesche (Ed.), 
ISBN ----, e-ISBN ----

Electrophoresis.
Theory and Practice
Michov, 
ISBN ----, e-ISBN ----

Pharmaceutical Chemistry.
Vol : Drug Design and Action
Vol : Drugs and Their Biological Targets
Campos Rosa, Camacho Quesada, 
Set: ISBN ----
Lars Backman

Protein Chemistry
Author
Professor Lars Backman
Umeå University
Department of Chemistry
Biochemistry
SE-901 87 Umeå
Sweden
[email protected]

ISBN 978-3-11-056616-1
e-ISBN (PDF) 978-3-11-056618-5
e-ISBN (EPUB) 978-3-11-056628-4

Library of Congress Control Number: 2019947529

Bibliographic information published by the Deutsche Nationalbibliothek

The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie;
detailed bibliographic data are available on the Internet at http://dnb.dnb.de.

© 2020 Walter de Gruyter GmbH, Berlin/Boston

Cover image: (c) Lars Backman
Typesetting: Integra Software Services Pvt. Ltd.
Printing and binding: CPI books GmbH, Leck

www.degruyter.com
Preface
This textbook is intended for students and those who need an intelligible text on
proteins, their structures and functions. Although the text is intended for students
with a basic knowledge of general chemistry, I believe also those with a limited
chemical cognizance would benefit by reading it.
The opening chapters give a short account of the cellular organization and the
fundamental concepts that govern chemical systems and reactions. Since these con-
cepts are universal, the same set of rules also governs the inner workings of the cell
in any organism and consequently all biological life.
The next three chapters present the players (i.e., the building blocks) and their
properties and structures as well as how they interconnect to form teams and larger
building blocks. This is followed by several chapters discussing proteins from dif-
ferent aspects. One chapter is devoted to the cellular workhorses, the enzymes that
make sure that a certain reaction occurs and that it occurs at the right time.
The book ends with a chapter on protein purification, which should prime the
reader for practical work with proteins and a chapter giving a short introduction to
structure determination of proteins.
There are many images in the book that display proteins but the format makes
it difficult to appreciate the images fully. Therefore, I suggest the reader to down-
load the protein structure file from RCSB Protein Data Bank (www.rcsb.org) and use
a molecular viewer to display the structure on a computer screen. A good and easy-
to-use molecular viewer is the freely available UCSF Chimera. All protein structures
in the book have been produced with Chimera.
The content of this book is based on a 9-week course I have given for several
years to students enrolled in a master’s program in biotechnology at Umeå
University during their second year of studies.
I am also thankful to Oleg Lebedev at de Gruyter, who talked me into writing
this book, and to Lena Stoll and Ria Fritz who succeeded him and has turned my
text into something that reads smoothly.
Many thanks to my colleagues Per-Olof Westlund, Magnus Andersson, Magnus
Wolf-Watz, Tobias Sparrman, Karina Persson, Michael Hall and Mikael Oliveberg,
who all have read and given me feedback on various parts of the text, which un-
doubtedly have improved the text. Special thanks go to my wife Anna, who has
pushed and inspired me all along the road to reach the final full stop.

Lars Backman
Umeå, August 2019

https://doi.org/10.1515/9783110566185-202
Contents
Preface V

1 The ballpark where it all occurs 1

1.1 The tree of life 2
1.2 Bacteria 3
1.3 Archaea 4
1.4 Eukaryotes 4
1.4.1 Nucleus 4
1.4.2 Endoplasmatic reticulum 4
1.4.3 Golgi apparatus 5
1.4.4 Mitochondria 6
1.4.5 Lysosome 7
1.4.6 Peroxisome 7
1.4.7 Chloroplast 7
1.4.8 Vacuol 9
1.4.9 Cytoplasm, cytosol and cytoskeleton 9
1.5 Water 9
Further reading 12

2 The rules 13
2.1 The hydrophobic effect 13
2.2 Covalent bonds 13
2.3 Noncovalent bonds 14
2.3.1 Ion–ion interactions 15
2.3.2 Hydrogen bonds 15
2.3.3 Van der Waals interactions 17
2.4 pH 18
2.5 Numbers are important 18
Further reading 19

3 The players 21
3.1 Twenty different α-amino acids 23
3.1.1 Nonpolar amino acids 24
3.1.2 Polar-uncharged amino acids 26
3.1.3 Polar-charged amino acids 27
3.1.4 Aromatic amino acids 29
3.1.5 Substitutes 30
Further reading 32
VIII Contents

4 The team 33
4.1 The peptide bond 33
4.2 The Ramachandran graph 36
4.3 The primary structure 37
4.4 The secondary structure 39
4.4.1 α-helix 39
4.4.2 β-Strands and β-sheet 43
4.4.3 Turns and loops 46
4.5 Secondary structure prediction 48
4.6 The tertiary structure 48
4.7 The quaternary structure 51
Further reading 52

5 The league 53
5.1 More water 54
5.2 Domains 55
5.2.1 How to acquire a domain 59
5.2.2 Classifying domains 61
5.3 Motifs 64
5.4 Topology and connectivity 66
5.5 Why should proteins be grouped? 70
Further reading 71

6 El clásico 73
6.1 Myoglobin 73
6.2 Hemoglobin 75
6.2.1 The role of heme 77
6.2.2 The Bohr effect 78
6.2.3 2,3-Bisphoshoglycerate 81
6.2.4 Fetal hemoglobin 82
6.2.5 Sickle-cell diseases and sickle-cell anemia 82
6.2.6 Allostery and cooperative binding 83
Further reading 85

7 The ball is round 87

7.1 Collagen 87
7.2 Fibroin – a valuable protein 90
7.3 Coiled coil and helical bundles 93
7.4 Fibrous structures 96
7.5 Microtubules 99
7.6 Actin filaments 103
Further reading 109
 Contents IX

8 The midfield 111

8.1 Lipid bilayers 111
8.2 Membrane proteins 113
8.3 Glycophorin 114
8.4 The fluid mosaic membrane model 117
8.5 Integral membrane proteins 119
8.6 The hydropathy index 122
8.7 The signal peptide 123
Further reading 125

9 The playmaker 127

9.1 A little bit of thermodynamics 127
9.2 The biological workhorse 129
9.3 Reaction rate 130
9.4 The steady-state assumption 131
9.5 The Michaelis–Menten constant 134
9.6 The active site 137
9.7 Lysozyme 138
9.8 Serine proteases 141
9.9 Enzyme control 145
9.9.1 Feedback inhibition 149
9.9.2 Alloster control 149
9.9.3 Covalent modification 150
9.9.4 Proteolytic control 152
9.9.5 Regulatory proteins 155
9.9.6 Synthesis control 157
9.10 Enzyme classes 157
Further reading 159

10 The striker 161

10.1 Levinthal’s paradox 161
10.2 The energy landscape theory and foldons 162
10.3 Why do proteins fold? 164
10.4 In vivo folding 165
10.5 Proteins with two distinct structures 167
10.6 Intrinsic disordered proteins 168
10.7 Moonlighting 169
Further reading 172

11 Full-time 173
11.1 Rotary motors 173
11.1.1 Bacterial flagellar motor 177
X Contents

11.2 Muscle contraction 178

11.2.1 Myosin 179
11.2.2 The motor domain 180
11.2.3 The cross-bridge cycle 182
11.2.4 Role of calcium ions in skeletal muscle contraction 183
11.2.5 Myosin domain architecture 183
11.2.6 Myosin-dependent cargo transport 186
11.3 Transport along microtubules 187
11.3.1 Dynein 187
11.3.2 Kinesin 190
11.4 Nonmotile P-loop NTPase 197
Further reading 200

12 Extra time 203

12.1 Buffers 203
12.2 Preparation of extract 205
12.3 Solubility and precipitation 206
12.4 Chromatography 208
12.4.1 Ion-exchange chromatography (IEX) 209
12.4.2 Hydrophobic interaction chromatography (HIC) 212
12.4.3 Affinity chromatography (AC) 213
12.4.4 Reverse-phase chromatography (RFC) 215
12.4.5 Gel filtration (GF) 215
12.5 Protein purification in a nut shell 217
12.6 Where is the protein? 218
12.6.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE 219
Further reading 222

13 Penalty shoot-out 223

13.1 X-ray crystallography 223
13.2 Nuclear magnetic resonance (NMR) spectroscopy 225
13.3 Cryogenic electron microscopy (cryo-EM) 227
13.4 What does the protein structure look like? 227
Further reading 229

Index 231
1 The ballpark where it all occurs

The minimal functional unit of any living organism is the cell. Inside this confined
space, the machinery required to produce everything necessary to survive (sustain life)
is present. The presence of genetic material allows each cell to divide and thereby pro-
duce a progeny. Cells come in all shapes as well as sizes; some are extreme like some
human neurons that can be close to a meter long but most are rather small, with diam-
eters around 10 μm or less. The number of cells in an organism also differs, from a
single cell in bacteria to some 1013–1014 cells in adult human (Figure 1.1).

Figure 1.1: Some typical cell shapes showing a bacteriophage, an endothelial cell, a nerve cell, a
fibroblast and a red blood cell.

Irrespective of shape or size, all cells have certain common characteristics. They are
all surrounded by a barrier, a membrane (sometimes called plasma membrane),
separating the interior of the cell from the exterior. In the membrane, there are usu-
ally “doors” or “ports” that allow import as well as export of molecules and ions.
Some molecules can diffuse through the membrane, whereas others require intri-
cate systems to pass the barrier. There are receptors on the exterior surface of the
membrane that accept external signals and transmit them across the membrane, to
an internal acceptor, that in turn activates an intracellular process. Similar mecha-
nisms are used to transmit signal the opposite way, from the inside of the cell to its
outside. The transport across the membrane as well as all other processes occurring
in the cell relay on proteins.
The number of proteins differs greatly from organism to organism. One of the
smallest organisms, the parasite Mycoplasma genitalium (causes urethritis), contains
482 protein-coding genes, but only 382 of these genes are essential for survival. The
genome of the endosymbiont Candidatus Carsonella ruddii is even smaller and codes
for 182 proteins. This is in contrast to the human genome that consists of ca. 20,000 –
21,000 protein-coding genes that due to alternative splicing may give rise to many
more proteins (more than one protein with different functions). The human genome
also contains some 16,000–21,000 noncoding genes.

https://doi.org/10.1515/9783110566185-001
2 1 The ballpark where it all occurs

1.1 The tree of life

It is believed that life on Earth occurred about 3.7–4 billion years ago. In spite of
tremendous efforts, the nature of the initial life is unknown, but it can be assumed
that it was very simple. With time, functions were gained and evolved and life de-
veloped into something that constitutes the common ancestor to life of today.
This common ancestor gave rise to two branches in the tree of life. One branch
that includes all bacteria and the other one that branched off with time into two
separate branches: archaea and eukaryotes. During evolution, each of these three
major groups have evolved and given rise to numerous organisms. It is estimated
that there may be up to 1012 different bacterial and archaeal species on Earth.
At the top of the tree of life, animals are located, thus being very recent inven-
tions. For instance, the first human population diverged only about 300,000 years
ago in Africa (Figure 1.2).

Eukaryotes
Bacteria Red algea
Green Plants Green algea Slime molds Animals Fungi
Purple bacteria filamentous
Gram bacteria
positives Archaea Alveolates
Halobacteria
Cyanobacteria
Thermophilic and
acidophilic archaea Stramenopiles

Entamoeba

Flagellates
Chlamydiae Trypanosoma
Flavobacteria
Trichomonads
Green sulfur bacteria
Spirochaetes
Diplomonads
Thermotoga

Aquifex

Origin of life

Figure 1.2: The tree of life.

 1.2 Bacteria 3

1.2 Bacteria

Bacteria can be divided into three groups depending on their shape: spherical (coc-
cus), rod-like (bacillus) and curved (vibrio, spirillum or spirochete). Although bac-
teria are unicellular, they may communicate with each other when forming biofilms
through quorum sensing by using pheromones.
Bacteria are generally very small. The rod-shaped Escherichia coli is about 2 μm
long and 0.5 μm in diameter. However, there are bacteria that are smaller and some
are much larger.
Bacteria, like all other organisms, are surrounded by a membrane that controls
the flow of molecules, prevents the loss of cell constituents and maintains proper
intracellular milieu. The membrane is covered by a rigid cell wall or envelope,
made of a peptidoglycan. In Gram-positive bacteria, the peptidoglycan is very thick
and retains the Gram stain. The peptidoglycan layer in Gram-negative bacteria is
very thin and therefore does not retain the Gram stain (Figure 1.3).

Gram negative Gram positive

Outer membrane

Peptidoglycan
Periplasmic space
Plasma membrane

Plasma membrane

Cell wall
Ribsome

Flagella
Pili DNA
Cytosol

Figure 1.3: A typical bacterial cell.

The bacterial genome is localized to the nucleoid that often contains a single circu-
lar DNA molecule. The genome of E. coli consists of ca. 4,600,000 bp that under
proper growth conditions are translated to proteins by more than 50,000 ribosomes.
On the surface of the envelope, there are flagella and pili. Flagella allow the cell to
swim in solution and pili provide adhesion points to the surface of animal cells.
4 1 The ballpark where it all occurs

1.3 Archaea

Archaea are the only organisms that inhabit extreme environments, such as hot
springs, extremely acidic or alkaline waters. A well-studied archaeon is Halobacterium
that thrives in extremely saline solution.
The shapes of archaea are similar to bacteria. Both archaea and bacteria move
by means of flagella and divide by binary fission. The cell wall of archaea lacks pep-
tidoglycan and the lipids of the membrane are different as they contain hydrocar-
bons rather than fatty acids that are linked to the glycerol moiety by ether bonds
and not ester bonds as in bacterial and eukaryotic membrane lipids. It is also evi-
dent that metabolic pathways in archaea are distinct.
Based on genomic and biochemical studies, it is suggested that archaea are closer
related to eukaryotes than to bacteria. In fact, it is not clear whether archaea and eukar-
yotes had a common ancestor or that the first eukaryote arose directly from an archaeon.

1.4 Eukaryotes

Eukaryotes are distinguished from bacteria and archaea by the presence of mem-
brane-bounded organelles. In particular, the genetic material is contained within a
distinct nucleus enclosed by a nuclear membrane or envelope. Eukaryotes can be
classified as either animals, plants, fungi or protists. Any organism with a nucleus
that are not animal, plant or fungus are classified as protists. Another classification
separates eukaryotes into Excavate, Chromalveolata, Rhizaria, Archaeplastida and
Unikonts. In this classification, animals and fungi, along with some protists, are
placed into Unikonta and plants into Archaeplastida, whereas Excavate,
Chromalveolata and Rhizaria include all other protists (Figure 1.4).

1.4.1 Nucleus

Most of the genetic material in a cell is contained in the cell nucleus. The nucleus is sur-
rounded by the nuclear envelope that consists of the outer and inner nuclear mem-
branes. In the envelope, there are nuclear pores that allow transport of molecules in
both directions across the envelope. The nucleolus is a region of the genetic material
that is particularly active and coding for ribosomal proteins and RNAs.

1.4.2 Endoplasmatic reticulum

The endoplasmatic reticulum or ER is an interconnected network of tubular mem-

branes and flattened sacs, also called cisternae. The interior of the ER constitutes
 1.4 Eukaryotes 5

Plasma membrane
Golgi apparatus

Ribosome

Rough endoplasmatic
reticulum Peroxisome

Nucleolus Mitochondria

Nucleus

Lysosome

Figure 1.4: A general animal cell with cell organelles.

the lumen. As the ER is continuous with the outer nuclear membrane, the lumen
and the volume between the two nuclear membranes are connected. The outer sur-
face of the rough ER is studded with ribosomes, the site of protein synthesis.
Proteins synthesized by ribosomes attached to the rough ER are generally destined
for membranes or for export via the Golgi apparatus. Soluble proteins, present in
the cytoplasm or in cell organelles, are often synthesized by free ribosomes.
The smooth ER has no ribosomes attached and is therefore not involved in pro-
tein synthesis. However, the smooth ER is important for the production of lipids
and steroids (Figure 1.5).

1.4.3 Golgi apparatus

Similar to the ER, the Golgi apparatus consists of a series of membrane sacs. Named
after the Italian discoverer Camillio Golgi, the Golgi apparatus is the site for packag-
ing and modification (primarily adding carbohydrates) of proteins that are to be ex-
ported out of the cell. Proteins destined for export initially accumulate in the ER
lumen before budding of in a vesicle that will fuse with the Golgi apparatus, where
it will be processed further and packed in a secretory vesicle.
6 1 The ballpark where it all occurs

Golgi apparatus

Ribosome
Rough
endoplasmatic
reticulum

Nucleolus

Nucleus

Figure 1.5: Nucleus, endoplasmatic reticulum and Golgi apparatus.

1.4.4 Mitochondria

The mitochondria are important for energy conversion. Oxidation processes, such as
glycolysis, citric acid cycle and fat oxidation, cause reduction of NAD+ and FAD to
NADH and FADH2, respectively. Reduced NADH and FADH2 are oxidized (and can be
reused) when electrons are transferred to acceptor complexes in the electron transport
chain. As electrons are transported along the chain and finally reduce molecular oxy-
gen, protons are transferred from the interior of the mitochondria to the space between
the outer and inner mitochondrial membrane. The proton gradient that arises is then
used to fuel the ATP synthase to generate ATP from ADP and inorganic phosphate.
Together these two processes, electron transport and oxidative phosphorylation, con-
stitute cellular respiration.
The inner mitochondria membrane is folded, creating cristae. Most of the com-
ponents of the electron transport chain are located in or on the cristae. The semi-
fluid matrix constitutes the interior of the organelle. It is in the matrix that the
reactions of the citric acid cycle and fat oxidation occur.
A small amount of genetic material as well as all components required to transcribe
and translate the genetic information is also present in the mitochondria (Figure 1.6).
 1.4 Eukaryotes 7

Inner mitochondria
membrane
Cristae

Matrix
Outer mitochondria
membrane Figure 1.6: A mitochondrion.

1.4.5 Lysosome

All eukaryotic cells are equipped with a waste disposal site. The lysozyme contains
enzymes that can degrade any biological molecules. Therefore, it is important that
the activities of these enzymes are confined inside the lysosome. This is accom-
plished in two ways. First, lysosomal enzymes are processed through the ER and
Golgi apparatus and transported to the lysosome sequestered in vesicles. Secondly,
the acidic milieu of the lysosome (pH < 5) is required for full activity, whereas in the
neutral cytoplasm (pH 7.2) the activity would be lower.

1.4.6 Peroxisome

Like the lysosome, the peroxisome is enclosed by a single membrane and also
about of the same size. The peroxisome has an important role in breaking down
long chain fatty acids to medium chain fatty acids that can be processed further by
the mitochondrion. The peroxisome is also important for the detoxification of hy-
drogen peroxide as well as other toxic substances, such as ethanol. In plant cells,
specialized peroxisomes called glyoxysomes have a prominent role in converting
fatty acid to carbohydrates during seed germination (Figure 1.7).

1.4.7 Chloroplast

In contrast to animal cells, plant cells contain chloroplasts and vacuoles. Like the mito-
chondrion, the chloroplast is surrounded by a double membrane and inside the chloro-
plast there is a membrane system called the thylakoid membrane. The thylakoid
8 1 The ballpark where it all occurs

Golgi apparatus

Ribosome

Nucleolus

Nucleus

Chloroplast
Rough
endoplasmatic
reticulum

Plasma
membrane
Mitochondria

Cell wall
Vacuol

Figure 1.7: Schematic drawing of a plant cell.

membrane is the site of photosynthesis, where sunlight is captured and used to produce
oxygen from water and at the same time converting the trapped energy into chemical
useful energy in the form of ATP and NADPH that is used for carbon fixation.
The compartment between the inner chloroplast membrane and the thylakoid
membrane is called stroma and that inside the thylakoid membrane is the thylakoid
lumen. The thylakoid membrane form grana stacks that are connected by stroma
thylakoids.
Some of the proteins required for photosynthesis and carbon fixation are syn-
thesized by the chloroplast itself (Figure 1.8).

Inner and outer membranes

Grana stack

Stroma

Thylakoidmembrane

Figure 1.8: Schematic drawing of a chloroplast.

 1.5 Water 9

1.4.8 Vacuol

Plants usually contain a single vacuole that grows with time. The plant vacuole is a
storage site for waste products, such as phenols and waxes. The vacuole maintains
the turgor pressure that keeps the plant from wilting.

1.4.9 Cytoplasm, cytosol and cytoskeleton

The volume inside a cell, with organelles and everything else present in the cell, is
called the cytoplasm. The soluble part of the cytoplasm that is the compartment be-
tween the plasma membrane and all cell organelles constitutes the cytosol.
The cytosol contains an intricate network of proteins called the cellular cytoskel-
eton. This network, composed to varying degrees of actin filaments, intermediate fila-
ments and microtubules, are essential for many cellular processes (Figure 1.9).

Figure 1.9: The cytoskeleton of a fibroblast. This is one of the first images showing the protein
network or cytoskeleton in a cell. Blue indicates the distribution of actin, the main protein of
microfilaments. Red marks the distribution of the actin-binding protein vinculin, a protein that
anchors filamentous actin to the membrane. Green shows the distribution of tubulin, the main
protein of microtubule. Courtesy of Victor Small, Austrian Academy of Science. From “The molecules
of the cell matrix” by Klaus Weber and May Osborne (1985) Scientific American 253: 92–102.

1.5 Water

Water is the most abundant substance in all living organisms, making up more than
70% of the body weight. The presence of water creates a fluid environment that allows
10 1 The ballpark where it all occurs

a molecule to move and therefore to interact with other molecules. The chemical prop-
erties of water also make it an excellent solvent.
The electronegative oxygen atom in a water molecule attracts the hydrogen
electrons, giving the oxygen partial negative charge and the two hydrogen atoms a
partial positive charge. This makes the water molecule a dipole, with one side nega-
tively charged and the other positively charged. Therefore, a water molecule can
interact electrostatically with other water molecules as well as with other polar mol-
ecules and thereby form hydrogen bonds (Figure 1.10).

δ+

104.5°

O
δ−
δ+
H

Lone pair
δ−

Figure 1.10: The arrangement of bonds and lone pairs can be described as a distorted tetrahedral.
The lone pairs affect the hydrogen atoms and push them closer to each other. In a perfect
tetrahedral arrangement, all angles should be 109.5 °.

A water molecule can form four hydrogen bonds – the two hydrogen atoms as do-
nators and the two lone pair electrons as acceptors. However, the actual number of
hydrogen bonds depends on the state of water. Water molecules in ice are perfectly
coordinated, as in other solids, and each water molecule has hydrogen bonds to
four neighboring molecules. In water, thermal molecular motion causes hydrogen
bonds to break, thereby decreasing the average number of hydrogen bonds of each
molecule. The perfect coordination is lost. Up to a certain temperature, which hap-
pens to be 4 °C, the molecules are more closely packed. At higher temperatures,
thermal motion leads to an expansion; the water “swells.” A very important conse-
quence of this behavior is that the density of water is greater than that of ice. This is
the reason why ice floats on water (Figure 1.11).
The anomaly properties of water are also due to hydrogen bonding. Compared
to other hydrides, the melting and boiling points of water are around 100 °C higher
than expected (Figure 1.12).
The high surface tension and viscosity of water is also due to hydrogen bond-
ing. The capillary force that allows plants to bring water up through the root system
 1.5 Water 11

Figure 1.11: In hexagonal ice, the water molecules are perfectly coordinated, each water molecule is
hydrogen bonded to four other water molecules. When ice melts, the perfect coordination is lost
and each water molecule is no longer bound to four other molecules.

100

50
Temperature °C

H2O H2Te
0
H2Se
H2S
–50

–100
0 50 100 150
Molar mass

Figure 1.12: Melting and boiling points of hydrides. The normal behavior for atoms in the same
group in the periodic system is that the melting and boiling points would increase with molar
mass. The dotted line indicates the expected melting and boiling points of water.

is dependent on the surface tension of water. The large heat capacity of water is
also related to the ability to form hydrogen bonds.
The polar property makes water an excellent solvent for other polar substances.
Thus, any substance that forms ions in solution, such as a salt like sodium chloride,
or can form hydrogen bonds can be dissolved in water (Figure 1.13).
Water is indeed a remarkable solvent!
12 1 The ballpark where it all occurs

+
+
– –
+ +
+

+
–

+
+
–

–
+

+
–

+
–
+

+ +
+ + –
+

+
+

+
– +
–
–

–
+
+

+
+ + –
– + +

Figure 1.13: Solvation of sodium chloride in water. When sodium chloride dissolves in water, the
water molecules form a shell around each ion, turning the partial negative oxygen toward the
sodium ion and the positive hydrogen toward the negative chloride ion.

Further reading
Bakshi, S., Siryaporn, A., Goulian, M. and Weisshaar, J.C. (2012). Superresolution imaging of
ribosomes and rna polymerase in live Escherichia coli cells. Mol Microbiol 85:21–38.
Baum, D.A. and Baum, B. (2014). An inside-out origin for the eukaryotic cell. BMC Biol 12:76.
Eme, L., Spang, A., Lombard, J., Stairs, C.W. and Ettema, T.J.G. (2017). Archaea and the origin of
eukaryotes. Nat Rev Microbiol 15:711–723.
Ezkurdia, I., Juan, D., Rodriguez, J.M., Frankish, A., Diekhans, M., Harrow, J., Vazquez, J., Valencia,
A. and Tress, M.L. (2014). Multiple evidence strands suggest that there may be as few as
19,000 human protein-coding genes. Hum Mol Genet 23:5866–5878.
Forterre, P. (2013). The common ancestor of archaea and eukarya was not an archaeon. Archaea
2013:372396.
Glass, J.I., Assad-Garcia, N., Alperovich, N., Yooseph, S., Lewis, M.R., Maruf, M., Hutchison, C.A.,
3rd, Smith, H.O. and Venter, J.C. (2006). Essential genes of a minimal bacterium. Proc Natl
Acad Sci U S A 103:425–430.
Locey, K.J. and Lennon, J.T. (2016). Scaling laws predict global microbial diversity. Proc Natl Acad
Sci U S A 113:5970–5975.
Nilsson, A. and Pettersson, L.G. (2015). The structural origin of anomalous properties of liquid
water. Nat Commun 6:8998.
Plopper, G., Sharp, D., Sikorsk, E. and Lewin, B. (2015). Lewin’s cells. Jones and Bartlett Publishers.
Sudbury, Mass.
Sharp, K.A. (2001) “Water: Structure and properties.” Encyclopedia of Life Sciences DOI:
doi: 10.1038/npg.els.0003116.
Tamames, J., Gil, R., Latorre, A., Pereto, J., Silva, F.J. and Moya, A. (2007). The frontier between cell
and organelle: Genome analysis of candidatus carsonella ruddii. BMC Evol Biol 7:181.
Williams, T.A., Foster, P.G., Cox, C.J. and Embley, T.M. (2013). An archaeal origin of eukaryotes
supports only two primary domains of life. Nature 504:231–236.
2 The rules

For a spontaneous process, the change in Gibbs free energy (ΔG) is always negative
(is less than zero). Changes in both enthalpy (ΔH) and entropy (ΔS) contribute to
Gibbs free energy as ΔG = ΔH − TΔS, where T is the absolute temperature in Kelvin.
The change in enthalpy is related to breaking and forming bonds, whereas entropy
can be seen as a measure of disorder.
The second law of thermodynamics states that the entropy of an isolated system
will increase over time for a spontaneous process. If we consider protein folding,
obviously a spontaneous process, the unfolded protein is highly disordered in con-
trast to the fully folded and functional protein; the entropy decreases. This appears
to violate the second law of thermodynamics. However, we need to consider that
the folding occurs in water.

2.1 The hydrophobic effect

Proteins contain both polar and nonpolar groups (i.e., amino acid residues). A polar
group, such as a carboxyl or amino group, can form hydrogen bonds with water mol-
ecules as well as with other polar groups. On the other hand, nonpolar groups cannot
form hydrogen bonds and cause a disturbance in the water structure. Therefore,
water molecules form a cage-like structure around nonpolar groups; these water mol-
ecules are ordered and the entropy decreases. During the folding process, these non-
polar groups are removed from contact with water by being placed in the interior of
the fully folded protein, “releasing” the caged water and avoiding further contact
with the surrounding water, thus increasing the disorder of the water molecules and
therefore also the entropy.
As protein folding occurs spontaneously, the entropic contribution from the be-
havior of water (ΔSwater) must be larger than that from to the actual folding process
(ΔSprotein); therefore, we can express the change in entropy as ΔS = ΔSwater − ΔSprotein
and ΔS must positive (be larger than zero; Figure 2.1).
This effect, called the hydrophobic effect, is the driving force for protein folding as
well as for membrane formation. The hydrophobic effect implies that without nonpolar
groups or as it is usually called a hydrophobic core a protein will not be able to fold.

2.2 Covalent bonds

Covalent bonds are formed when two atoms share electron pairs. The peptide bond,
the covalent bond formed between two amino acids during protein synthesis, is
strong and static; once it is formed nothing more can happen.

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14 2 The rules

Water molecule Nonpolar group

Figure 2.1: Protein folding places nonpolar inside the protein, without contact with the surrounding
polar water, thereby increasing the entropy, and driving the folding process.

Many cellular processes require transient contacts between proteins or be-

tween a protein and a small molecule (ligand). Initially, the interacting molecules
are juxtaposed, bonds are formed to keep them attached to each other for a cer-
tain time and then the bond(s) need to be broken to allow the complex to dissoci-
ate. From an energy point of view, covalent bonds are costly to break or make; the
energy required to break a single carbon–carbon (346 kJ/mol) or oxygen–carbon
(358 kJ/mol) bond is about 350 kJ/mol. Therefore, covalent bonds are not well
suited to stabilize molecules that undergo continuous conformational changes or
to keep components attached and stabilize short-lived complexes due to the high
energy cost.
Noncovalent bonds are much better suited for dynamic purposes.

2.3 Noncovalent bonds

It may be surprising but proteins are not very stable structures. Many proteins are
stable by only 40–50 kJ/mol. Since the energy required to break a single hydrogen
bond is about 20 kJ/mol, it may be enough to break a few weak bonds to unfold or
denature a protein and make it nonfunctional. It is important to realize that this
does not mean that there are only a few noncovalent bonds in a protein, but rather
that the energy gain by forming intramolecular hydrogen bonds compared to hydro-
gen bonds with water is small.
 2.3 Noncovalent bonds 15

2.3.1 Ion–ion interactions

A permanent ion may attract an ion of opposite charge and form a salt bridge.
Although ions of the same charge will not form a bond, the repulsive force can still
be important.
According to Coulomb’s law the force F between two charged ions, with charges
q1 and q2 Coulombs (C), separated by a distance r is defined as
q1 · q2
F=k·
r2

where k is Coulomb’s constant, equal to 9·109 J·m·C−2 in vacuum. Since the force
depends on the medium, Coulomb’s constant can be rewritten to give
1 q1 · q2
F= ·
4πε0 εr ðT Þ · r2
1 q1 · q2
F= ·
4πε0 κ · r2

where ε0 is the permittivity in vacuum. εr(T) is the relative permittivity and κ is the
dielectric constant that reflects what effect the medium has on the force. The dielec-
tric constant is strongly influenced by the nature of the medium. In a nonpolar sol-
vent, such as benzene or hexane, the dielectric constant is 2–3, whereas in a polar
solvent like water the dielectric constant is around 80. For an ion pair separated by
4 Å, the energy of interaction will be −39 kJ/mol in water, whereas in a nonpolar
solvent it would be nearly 40 times larger. In other words, electrostatic interactions
are weakened by polar media.
In water, a solvated sodium ion will attract not only a negatively charged chloride
ion but also water molecules. The water dipole will reorient in such a way that the par-
tially negatively charged oxygen will face the positive sodium ion and the partially pos-
itively charged hydrogen will face negative chloride ion. This polarization creates a
layer of water molecules surrounding the ions, causing a screening effect that reduces
the Coulomb interaction between the sodium and chloride ions. In a nonpolar medium,
there are no solvent molecules that can interact with the ions and the charges are not
screened, and there are no favorable interactions with the medium (Figure 2.2).

2.3.2 Hydrogen bonds

A hydrogen bond forms between a hydrogen donor and an acceptor. In proteins and
other biomolecules, the hydrogen donor is usually an electronegative nitrogen or
16 2 The rules

+ +
– +
+
+ –
–

+
+ + +
– +

+
–

–
+
+ +

–
+ + – +
–

+
+
+

+
+

+
– +
+

–
–

–
–

–
+
+

+
+
–

–
+

+ +
+ +

+
–
+

+
–

–
–
-

+
+

+
–
+
Figure 2.2: Screening of ion charges by layers of water dipoles. Dotted lines represent hydrogen
bonds.

oxygen with a covalent bound hydrogen. Fluorine may also participate in hydrogen
bonds, but is scarcely present in biomolecules. However, any atom that has a suffi-
cient electronegativity that induces a partial positive charge on the hydrogen atom
can form hydrogen bonds. The acceptor is usually an atom with lone pair of electrons
(such as nitrogen or oxygen), even though other fully or partially negatively charged
groups can function as an acceptor (Figure 2.3).

δ+ δ–
Donor-H : Acceptor

Hydrogen bond
: Acceptor
r-H
no
Do

Figure 2.3: An electronegative atom with a covalent bound hydrogen is the donor and an atom with
lone pair of electrons, such as nitrogen or oxygen, is the acceptor in a hydrogen bond.

The strength of a hydrogen bond depends on both geometry and distance of the
bond as well as donor atoms. The energy of interaction for a hydrogen bond with
nitrogens and/or oxygens is in the range of −10 to −40 kJ/mol and is highly depen-
dent on the solvent. The energy of a hydrogen bond in an α-helix in gas phase is
 2.3 Noncovalent bonds 17

around −23 kJ/mol, but only −8 kJ/mol in water. The reduction can be attributed to
the hydrophobic effect of water, involving contributions from the entropy. The
strength of a bond with C–H as donor is weaker (less than −10 kJ/mol and only
present in nonpolar environments). The distance between the donor atom and ac-
ceptor atom varies, but is usually ~3 Å; a shorter distance gives stronger bonds. If
the donor and acceptor are separated by less than ~2.5 Å and have similar pKas, the
energy required to transfer the hydrogen from donor to acceptor is very low. Such a
low barrier hydrogen bond is very strong and the strength is estimated to be close
to half of the strength of a covalent bond.
The geometry is also important. A linear hydrogen bond, where donor-H is in
line with the acceptor’s lone pair orbital is stronger than a bond where there is an
angle between the donor-H and acceptor (Figure 2.4).

H
O
C OH N O
C
H
OH

C O N P O
C N
H

Figure 2.4: Common functional groups in proteins that are important hydrogen bond donors or
acceptors.

2.3.3 Van der Waals interactions

A permanent dipole with its asymmetric distribution of electrons may attract an-
other dipole if they are close enough. Such a permanent dipole–permanent dipole
(or Keesom) interaction is weak (less than 3–4 kJ/mol) and highly dependent on the
distance and temperature. The energy of interaction, assuming that the dipoles are
constantly rotating, is averaged over all possible orientations of the dipoles and de-
creases with the inverse sixth power of distance:

B
EvdW = −
r6

where r is the distance between the dipoles and B is a positive constant of the at-
tractive force. The strongest van der Waals interactions occur when the two dipoles
are slightly more separated than the sum of their van der Waals radii.
18 2 The rules

A permanent dipole can induce a dipole moment in a closely placed molecule or

group that leads to a mutual electrostatic interaction. A permanent dipole–induced di-
pole interaction or Debye force is weaker than that between two permanent dipoles.
Motions of the electrons in one molecule may influence the electrons in another
molecule and induce a correlated motion. Such London dispersion forces depend
on random fluctuations of electrons. The strength of London dispersion forces is
weaker than that of Keesom and Debye forces.

2.4 pH

When an acid (HAc) is added to water, a fraction of the acid will dissociate and in-
crease the concentration of H+ (rather H3O+, a hydronium ion) and Ac−

HAc + H2 O!Ac − + H +

The equilibrium constant, Ka, is defined as

½Ac −  · ½H + 
Ka =
HAc

As pH is defined as

pH = − log½H + 

the increased concentration of H+ will lower the pH. The stronger the acid, the
larger the fraction that will dissociate and the lower the pH will be.
The strength of a weak acid is expressed by its pKa-value, which is defined as
the negative logarithm of the acid dissociation constant

pKa = − log10ðKa Þ

The relation between pH and pKa is given by the Henderson–Hasselbalch equation

½Ac − 
pH = pKa + log
½HAc

The expression signifies that the pH equals the pKa-value when exactly half of the
weak acid is dissociated (or ionized). In pure water that is considered as a neutral
solution, the concentration of H+ is 1·10−7 M due to autoprotolysis and therefore the
pH is 7. A solution with a lower pH is acidic, whereas a higher pH is basic.

2.5 Numbers are important

Proteins are mobile; they move around all the time in the cell. Some are recruited to
the plasma membrane, whereas others have to find a certain protein to interact
 Further reading 19

with and form a functional complex. Yet other proteins are activated or deactivated
by binding of small molecules. Thus, it is necessary that the two (or more) mole-
cules meet to form the complex. In the cell soup, proteins move back and forth in
three dimensions randomly. To form the functional complex, the two proteins have
to “collide” in the proper way to find the correct attachment points and bind to
each other. There are two main factors that will affect the success rate of these colli-
sions: number of molecules and diffusion rate.
Many proteins are present in the cell at concentrations around or less than
1 μM. Such a concentration corresponds to around ~6·1017 protein molecules per
liter. With this estimate, we can calculate the copy number of a protein within a
single cell. If we take a cell like the rod-shaped Escherichia coli that is about 2 μm
long and 0.5 μm in diameter, this cell has a volume close to 0.4 μm3 or 4·10−16 L. At
a protein concentration of 1 μM or less, we would expect to find less than 240 copies
of this particular protein. Obviously, the copy number of a certain protein at a mi-
cromolar concentration is rather limited.
The diffusion rate is more difficult to estimate due to the crowding effect. In an
E. coli cell, there are about 40,000 ribosomes and some 4,200 different proteins at
varying concentrations as well as numerous small molecules. Thus, it is rather obvi-
ous that the cell is packed with molecules that occupy space; the cell is undoubt-
edly crowded. This crowding probably reduces the diffusion rate of proteins and
other large molecules, whereas small molecules are probably not influenced to any
major degree.
Although crowding has a negative effect on the diffusion rate, it has at the
same time a positive effect; as the active concentration of any molecule will be
larger, any complex formation will be enhanced.

Further reading
Atkins, P. (2010). The laws of thermodynamics: A very short introduction. Oxford University Press.
Oxford.
Atkins, P., de Paula, J. and Keeler, J. (2018). Atkins’ physical chemistry. Oxford University Press.
Oxford.
Cleland, W.W. (2000). Low-barrier hydrogen bonds and enzymatic catalysis. Arch Biochem Biophys
382:1–5.
Milo, R., Phillips, R. and Orme, N. (2016). Cell biologt by the numbers. Garland Science, Taylor &
Francis Group, LLC. Abingdon.
Po, H.N. and Senozan, N.M. (2001). The Henderson-Hasselbalch equation: Its history and
limitations. J Chem Edu 78:1499–1503.
Sheu, S.Y., Yang, D.Y., Selzle, H.L. and Schlag, E.W. (2003). Energetics of hydrogen bonds in
peptides. Proc Natl Acad Sci U S A 100:12683–12687.
Steiner, T. (2002). The hydrogen bond in the solid state. Angew Chem Int Ed Engl 41:49–76.
3 The players

It is estimated that there are tens of thousands of different proteins in a human cell,
all performing different tasks. Still, they all are built in the same way from 20 sim-
ple molecules; 20 different α-amino acids.
All these 20 α-amino acids contain a carboxyl group, an amino group and a hy-
drogen atom, covalently bound to the so-called α-carbon. What distinguish an
amino acid from another is the side chain, R. If the side chain would be a methyl
group (–CH3), then this carbon would be the β-carbon (Figure 3.1).

COOH
{ Carboxyl
group

Amino
group } H2N Cα H

α-carbon
Side
chain } R

Figure 3.1: A typical α-amino acid.

However, when an α-amino acid is dissolved in water (or a neutral buffer) both the
amino and carboxyl groups ionize. As the carboxyl group is a weak acid, it will lose
a proton (deprotonated) and become negatively charged. In the same manner, the
amino group, being a weak base, will take up a proton (protonated) and become
positively charged. A molecule with both a negative and positive charge is called a
zwitterion. Although the amino acid is charged, its net charge is zero.
As long as the pH of the buffer is above the pKa of the carboxyl group and below
that of the amino group, both ionize and are charged. The carboxyl group will be pro-
tonated when pH drops below the pKa of the carboxyl group and the amino group
will be deprotonated when pH increases above the pKa of the amino group. The
charged of the amino acid will vary depending on the pH of the solvent (Figure 3.2).
All α-amino acids, except for glycine, are optically active, meaning that they
rotate plane-polarized light clockwise or counter-clockwise. An optically active mol-
ecule contains an asymmetric or chiral center, which is signified by a tetrahedral
carbon with four different substituents. Such a chiral molecule cannot be superim-
posed on its mirror image. As long as the side chain is anything else than a proton
(as in glycine), the α-carbon constitutes a chiral center (Figure 3.3).

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22 3 The players

COOH COO– COO–

+ +
H3N Cα H H 3N Cα H H2 N Cα H

R R R

acid acid base base

pH < pKa pKa < pH < pKa pKa < pH

Figure 3.2: The pH of the solvent will affect the ionization of the carboxyl and amino groups of the
amino acid. At pH < pKa of the carboxyl group, the carboxyl is protonated and uncharged and at
pH > pKa of the amino group, it is deprotonated and uncharged.

L-α-amino acid D-α-amino acid

Figure 3.3: L- and D-enantiomers of a typical α-amino acid.

The optical activity is related to the reference molecules L- and D-glyceraldehyde

(Figure 3.4). L-glyceraldehyde rotates plane-polarized light counter-clockwise and is
said to be levorotatory. D-glyceraldehyde is dextrorotatory as it rotates plane-polarized
light clockwise. Laevus and dexter are the Latin words for left and right, respectively.
Whether an amino acid is an L- or a D-enantiomer depends on the similarity to
glyceraldehyde. In a Fisher projection of glyceraldehyde, the hydroxyl group is placed
to the left in the L-enantiomer and to the right in the D-form. Therefore, when the
amino group is placed to the left, it is an L-α-amino acid. This does not necessarily
imply that an L-α-amino acid rotates plane-polarized light counter-clockwise. When
there are several chiral centers, the position of the hydroxyl (or analogous) group
furthest away from the aldehyde or keto group determines whether it is an L- or a
D-configuration. The D/L system is not very useful for describing the absolute configura-
tion of chiral centers. It should be remembered that α-amino acids present in proteins
are all in the L-configuration (Figure 3.5).
A convention to indicate the absolute configuration of chiral centers is based on
the “weight” or priority of the substituents at the chiral center. In this system, each
substituent is given a rank depending on the atomic number of the atom or group
 3.1 Twenty different α-amino acids 23

CHO CHO

HO C H H C OH

CH2OH CH2OH
L-glyceraldehyde D-glyceraldehyde

COO– COO–
+ + Figure 3.4: Fisher projections of glyceraldehyde and
H3N Cα H H Cα NH3 an α-amino acid. In the Fisher convention, horizontal
bonds are pointing out of the plane and vertical bonds
R R are pointing in to the plane. In the L-enantiomer, the
L-α-amino acid D-α-amino acid hydroxyl and amino group are to the left.

“CO” group

“N”
group
Figure 3.5: A simple mnemonic is the CORN rule. By
rotating the molecule, such that the hydrogen is in front
“R” group
of the α-carbon, reading CO-R-N in the clockwise direction
indicates an L-α-amino acid.

bound to the chiral center. If two substituents have the same priority, the atomic num-
bers of the next bound atoms are compared. The priority of some common groups
found in amino acids is: SH > OH > NH2 > COOH > CH2OH > CH3 > H. To determine
the absolute configuration, the priority of the four substituents is ranked from the
highest to the lowest and the molecule is oriented such that the group or atom
with the lowest priority is pointing away from the chiral center. If going from the
group with highest priority to the next in rank is clockwise, the chiral center has
an R-configuration (R for rectus, Latin for right). If the order is counter-clockwise,
the chiral center has an S-configuration (S for sinister, Latin for left). Thus, the α-
carbon in the L-α-amino acid in Figure 3.5 has an S-configuration. In fact, all but
one (cysteine) of the α-amino acid has an α-carbon in the S-configuration.

3.1 Twenty different α-amino acids

Since three of the substituents bound the α-carbon are the same, it is obvious that
different chemical and physical properties of each of the 20 α-amino acids depend
24 3 The players

on the side chain. The size of the side chain differs; the smallest side chain is only a
proton (–H). Some side chains contain a carboxyl or an amino group, and can be
ionized depending on the pH of the solvent. Some side chains are very nonpolar,
whereas others are polar, which will influence their solubility in water. Some con-
tain a conjugated ring system, giving them useful optical properties. Some side
chains are branched. The 20 amino acids are usually classified as nonpolar, polar-
uncharged, polar-charged or aromatic. It should be noted that some amino acids fit
in more than one group; a nonpolar-uncharged amino acid may have some polar
properties (Figure 3.6).

3.1.1 Nonpolar amino acids

The nonpolar amino acids are glycine (Gly; G), alanine (Ala; A), valine (Val; V), leu-
cine (Leu; L), isoleucine (Ile; I), methionine (Met; M) and proline (Pro; P). These
amino acids are characterized by an aliphatic side chain of varying length whose
hydrophobicity increases with increased chain length. The side chains of these
amino acids cannot participate in hydrogen bonding and have a propensity to
avoid contact with water. Therefore, the amino acids of this group are usually
found in the interior of proteins, shielded from water contact. Since the interior of a
protein is rather crowded, these side chains will be in close proximity to other side
chains and possibly participate in van der Waals interactions.
Glycine with only a hydrogen atom in the side chain can probably not partici-
pate in any type of interactions. However, the small size allows glycine to be placed
in regions where larger side chains do not fit. A branched side chain, as in valine,
leucine and isoleucine, is less flexible and has a larger packing sphere than a
straight hydrocarbon chain. It should also be noted that glycine is the only α-amino
acid with an achiral α-carbon.
The sulfur-containing side chain of methionine is considered as one of the most
hydrophobic side chains. Although the electron configuration of sulfur is similar to
that of oxygen, the electronegativity is much lower (2.58 compared to 3.44 for oxy-
gen) and close to that of carbon (2.55). The small difference in electronegativity be-
tween sulfur and carbon prevents the formation of a dipole. Despite methionine’s
nonpolar property, it can interact weakly with polar molecules and cationic metals,
such as iron, zinc and copper. Methionine is also important in the biosynthesis of
metabolic molecules such as taurine (present in bile) and S-adenosyl methionine
(donor of methyl groups in several metabolic processes).
Proline differs from the other amino acids, in that the side chain is covalently
bound to the α-amino group, forming a pyrrolidone ring. The cyclization reduces
the conformation space of proline; both the α-amino group and the side chain are
locked in space. This has important consequences for protein structure as will be
discussed below.
 Nonpolar
O O– O O– O O– O O– O O– O O– O O–
C C C C C C C
+ + + + + + +
H3N C H H3N C H H 3N C H H3N C H H3N C H H3N C H H2N C H
H CH3 CH CH2 HC CH3 CH2 H2C CH2
H3C CH3 CH CH2 CH2
C
H3C CH3 CH3 S H2

CH3
Glycine (Gly; G) Alanine (Ala; A) Valine (Val; V) Leucine (Leu; L) Isoleucine (Ile; I) Methionine (Met; M) Proline (Pro; P)

Polar-uncharged Aromatic
O O– O O– O O– – O O– O–O O O–
O O
C C C C C C C
+ + + + + + +
H3N C H H3N C H H 3N C H H 3N C H H3N C H H3N C H H3N C H
CH2 HC CH2 CH2 CH2 CH2 CH2
OH H3C OH C CH2 SH
H 2N O C
H2N O HN
Serine (Ser; S) Threonine (Thr; T) Asparagine (Asn; N) Glutamine (Gln; Q) Cysteine (Cys; C)
OH
Polar-charged Tryptophan (Trp; W) Tyrosine (Tyr; Y)
O O– O O– O O– OO– O O–
C C C C C O O–
+ + + + + C
H 3N C H H3N C H H3N C H H3N C H H3N C H
+
CH2 CH2 CH2 CH2 CH2 H3N C H
C CH2 CH2 CH2 +
C CH2
– HN CH
O O C CH2 CH2
– HC NH
O O CH2 NH
NH3+ C NH2+
NH2 Phenylalanine (Phe; F)
Aspartate (Asp; D) Glutamate (Glu; E) Lysine (Lys; K) Arginine (Arg; R) Histidine (His; H)
3.1 Twenty different α-amino acids

Figure 3.6: Twenty α-amino acids found in proteins in their ionized form at neutral pH. The structures common for all
amino acids are boxed in blue. The ionizations of aspartate, glutamate, arginine and histidine are assigned to a
single atom, but in reality the charges are delocalized.
25
26 3 The players

3.1.2 Polar-uncharged amino acids

Polar amino acids are soluble in water as they can interact favorably with the water
molecules. The hydroxyl group in the side chains of serine (Ser; S) and threonine
(Thr; T) and the amide group in the side chains of asparagine (Asn; N) and gluta-
mine (Gln; Q) form hydrogen bonds with water. Neither the hydroxyl group nor the
amide group is charged under most cellular conditions.
Both serine and threonine can undergo posttranslational modifications.
Phosphorylation of the hydroxyl by ATP (adenosine triphosphate) is a major mech-
anism in signal transduction as well as a control mechanism of enzyme activity
(Figure 3.7).

O
Ser CH2 OH + ATP Ser CH2 O P O– + ADP
OH

Figure 3.7: Phosphorylation of serine. In the enzymatic reaction a phosphate group from ATP is
transferred to the hydroxyl group of serine. When dephosphorylated, the hydroxyl-bound
phosphate is released as a free phosphate group; thus, ATP is not recovered.

Serine and threonine can also be glycosylated by attachment of a carbohydrate unit

to the hydroxyl oxygen forming an O-glycoside bond.
Under certain conditions, the hydroxyl group of serine participates in catalysis.
In a group of proteases (enzymes that digest proteins), called serine proteases, the
serine hydroxyl is activated by deprotonation, making a good nucleophile (–O−).
This only occurs when the serine hydroxyl is close to certain residues that facilitate
the deprotonation by lowering the pKa of the hydroxyl.
The amide groups of asparagine and glutamine can form hydrogen bonds, both
as hydrogen donator and acceptor. Similar to serine and threonine, asparagine and
glutamine can be glycosylated, but in this case the carbohydrate unit is linked by
an N-glycoside bond to the amide nitrogen.
The side chain of cysteine (Cys; C) contains a thiol group (or sulfhydryl). Due to
the electron configuration, sulfur has a partial negative charge, which makes the
side chain of cysteine slightly polar. Since it is the electron configuration and not
the electronegativity of sulfur that causes the partial charge, the hydrogen is not
affected as in a hydroxyl or carboxyl group. Thus, the thiol group is not involved in
hydrogen bonding.
The thiol group of cysteine undergoes redox reactions (Figure 3.8). Under oxi-
dizing conditions, cysteine can react with another cysteine in the proximity and
form a covalent bond or a disulfide bond, also called a disulfide bridge or S–S bond.
Since the intracellular milieu is reducing (due to the high concentration of NADPH
and glutathione), disulfide bonds are usually found in the interior of proteins.
 3.1 Twenty different α-amino acids 27

O O– O O–
C C
+H H +
3N C H3N C H
CH2 CH2
SH Oxidation
S
+
SH Reduction S

H 2C H2C
+ +
H C NH3 H C NH3
C C
–O
–O
O O
cysteine cystine Figure 3.8: The redox reaction of cysteine.

Cysteines located to the surface of proteins tend to be in the reduced state. During
purification of proteins, it is common to include a reducing agent in the media to
keep cysteines in the reduced state, avoiding the formation of covalent disulfide
bonds.

3.1.3 Polar-charged amino acids

The side chains of aspartate (Asp; D), glutamate (Glu; E), lysine (Lys; K), arginine
(Arg; R) and histidine (His; H) are ionized at neutral pH. This implies that they can all
participate in ion–ion interactions and in hydrogen bonds, as acceptors as well as do-
nators. Therefore, these residues are often found on the surface of proteins where they
can interact with other residues or with the surrounding water. Whenever a polar
(charged or uncharged) residue is located in the interior of a protein, it must have an-
other polar residue (or water molecule) close enough to form a hydrogen bond; other-
wise, the placement would be energetic unfavorable.
At neutral pH, the side chains of aspartate and glutamate are negatively
charged; they act as acids in that they release a proton. The side chains of lysine,
arginine and histidine are positively charged under the same conditions; they act
as bases in that they take up a proton.
The pKa values of the side chain carboxyls in aspartate and glutamate are
higher than those of the α-carboxyls. In addition, the pKa values of the side chain
amino groups of lysine and arginine are higher than those of the α-amino groups.
The reason being the different chemical surrounding in the side chains influence
the pKa values and thus the ability to be deprotonated or protonated (Table 3.1).
The side chain of arginine contains a guanidine group (or guanidinium group
in the protonated form). The three nitrogen atoms in the guanidinium group can all
28 3 The players

participate in ion–ion interactions as the charge is delocalized over the whole

group. The guanidinium group also has several possibilities to act as acceptor and
donor in hydrogen bond formation.

Table 3.1: Properties of α-amino acids.

Free amino acid Protein

Residue mass a
pKa–COOH b
pKa–NH b
pKa side chain b
pKa side chainc

Nonpolar
Glycine . . .
Alanine . . .
Valine . . .
Leucine . . .
Isoleucine . . .
Methionine . . .
Proline . . .
Polar-uncharged
Serine . . .
Threonine . . .
Asparagine . . .
Glutamine . . .
Cysteine . . . . . ± −.
Polar-charged
Aspartate . . . . . ± .
Glutamate . . . . . ± .
Lysine . . . . . ± .
Arginine . . . .
Histidine . . . . . ± .
Aromatic
Tryptophan . . .
Tyrosine . . . . . ± .
Phenylalanine . . .
a
The residue mass is the mass of the amino acid with an uncharged side chain in a protein. The
molecular mass of an amino acid is obtained by adding 18 (the mass of water) to the residue mass.
b
pKa values of free amino acids are from CRC Handbook of Chemistry and Physics, 73rd edition.
c
pKa values of some side chains when present in proteins are from G.R Grimsley, J.M. Scholtz and
N. Pace (2009) A summary of the measured pKa values of the ionizable groups in folded proteins,
Prot Sci 18:247–251.

The side chain amino group (ε-amino group) in lysine is rather reactive; it can form a
Schiff base with aldehydes and it can be oxidized as well as hydroxylated.
Histidine is the only amino acid with a pKa close to neutral pH; the pKa of histidine
is 6.0. Therefore, even relatively small changes in pH will change the average charge
(Figure 3.9). When the side chain is protonated, the positive charge is distributed
 3.1 Twenty different α-amino acids 29

between the two nitrogen atoms. When deprotonated, the hydrogen can be bound to ei-
ther of the nitrogen atoms in the planar ring. The imidazole group has aromatic proper-
ties independent on the protonation state and can form π-stacking interactions. In
contrast to the other aromatic amino acids (tryptophan, tyrosine and phenylalanine) his-
tidine does not absorb light in the near UV-region (280 nm) in any protonation state.

CH2
2 C
+
HN CH
HC NH

1
Net charge

0
2 4 6 8 10 12 14 pH

CH2
–1 C
N CH
HC NH

Figure 3.9: pH-titration of histidine (red square) and arginine (blue circle). The dashed line
indicates the charges at a pH equal to the pKas of the side chains. Insert: the side chain of
histidine in the charged form below pH 6 and in the uncharged form above pH 6.

3.1.4 Aromatic amino acids

The aromatic amino acids tryptophan (Trp, W), tyrosine (Tyr; Y) and phenylalanine
(Phe; F) all contain conjugated ring systems. There is an indole group in trypto-
phan, a phenol group in tyrosine and a benzene ring in phenylalanine. Since the
indole and phenol groups contain polar groups (NH and OH), the side chains of
tryptophan and tyrosine have some polar properties as well as nonpolar properties.
The aromatic ring systems are planar and the π-electrons are delocalized,
which give the ring system particular properties. The π-electrons will create a par-
tially negative charge above and below the ring, whereas the ring itself will be
partially positive. If two aromatic side chains are in proximity, either “face-to-face”
or “edge-to-face,” an attractive interaction, a π–π interaction, can be formed be-
tween the rings. The force is highly dependent on distance (it decays with r−6) and
geometry (it is stronger if the rings are not perfectly face-to-face but rather shifted
slightly). The perhaps best example of π–π interactions is the stacking of base
30 3 The players

pairs in DNA. In the DNA double helix, the distance between bases is only ~3.4 Å
and each base pair is rotated ~30°.
The side chain of histidine is not only polar and ionizable but also aromatic.
Thus, histidine can participate in π–π interactions. The partially negative charge of
the aromatic ring system can also form interactions with cations. In proteins, the
side chains of arginine and lysine can form cation–π interactions. It should also be
noted that the electronegative nitrogen atoms in the aromatic rings can function as
acceptors in hydrogen bonds.
Tryptophan, tyrosine and phenylalanine are important from a practical point of
view. Since they absorb light in the near UV region, their presence in proteins is
very useful to measure the concentration of proteins (Figure 3.10). The spectral
properties differ between the aromatic amino acids. Tryptophan absorbs ca. 5 times
more than tyrosine and ca. 50 time more than phenylalanine and the wavelengths
of their absorbance maxima are different. At 280 nm, the absorbance of histidine is
insignificant (Table 3.2).

6,000
Absorption coefficient

4,000

2,000

0
240 260 280 300 320
Wavelength (nm)

Figure 3.10: Absorption spectra of L-tryptophan (blue), L-tyrosine (red) and L-phenylalanine (black).
Data from M. Taniguchi and J.S. Lindsey (2018). Database of absorption and fluorescence spectra
of >300 common compounds for use in photochemcad. Photochem Photobiol 94:290–327.

3.1.5 Substitutes

In some proteins, certain amino acids are irreversibly modified after synthesis. In
collagen, a major component of fibrous tissues, such as cartilage, ligaments and
tendons, proline residues are modified to hydroxyl-proline in a reaction that re-
quires vitamin C and oxygen. If vitamin C is deficient, proline cannot be converted
 3.1 Twenty different α-amino acids 31

Table 3.2: Spectral properties of the aromatic amino acidsa.

Amino acid λmax nm Molar attenuation Relative absorbance

coefficientb M− ⋅ cm− at  nm

L-Tryptophan  , ±  .

L-Tyrosine  , ±  .
L-Phenylalanine . . ± . .
a
Data from E. Mihalyi (1968). Numerical values of the absorbance of the aromatic amino acids in
acid, neutral and alkaline solutions. J Chem Eng Data 13:179–182.
b
IUPAC recommends to use attenuation coefficient instead of extinction coefficient or molar
absorptivity.

to hydroxyl-proline, which ultimately leads to scurvy. Carboxylation of the γ-

carbon in the side chain of glutamate is important for the function of osteocalcin
(bone γ-carboxyglutamic acid-containing protein) in bone tissue and of several pro-
teins involved in blood coagulation. The formation of carboxyglutamate is depen-
dent on vitamin K (Figure 3.11).

O O–
C
+ C H
H3N
CH2
O O–
CH2
C O O–
+ C O O – CH2
H 2N C H
+ C H C
H 3N N CH2
H2C CH2 +
H 3N C H
CH2 C NH
C CH2 CH2 O
HO H SeH CH3
–OOC COO–

Hydroxy-proline γ-Carboxy-glutamate Seleno-cysteine (Ses; U) Pyrro-lysine (Pyl; O)

Figure 3.11: Common modified amino acids found in proteins.

Seleno-cysteine and pyrro-lysine are sometimes called the 21st and 22nd amino
acids as they are inserted in a few proteins during synthesis. However, neither are
coded directly by the genetic code; there is no codon that always translates to se-
leno-cysteine. Instead, seleno-cysteine is encoded by the UGA codon that normally
translates to a stop signal but due to a seleno-cysteine insertion sequence in the
mRNA and presence of selenium in the growth media, UGA is translated to seleno-
cysteine. In the absence of selenium, UGA translates as a stop and leads to a
32 3 The players

premature end of the protein synthesis. The pKa (5.47) and reduction potential of
seleno-cysteine are lower than that of cysteine. Seleno-cysteine is found in proteins
catalyzing redox reactions, such as glutathione peroxidases and thioredoxin
reductases.
Like seleno-cysteine, pyrro-lysine is also coded by a stop codon. In this case, it
is the “amber” stop codon UAG that codes for pyrro-lysine. Pyrro-lysine appears not
to be present in eukaryotic proteins, as it has been found only in some archaea and
bacteria.

Further reading
Barrett, G.C. and Elmore, D.T. (2008). Amino acids and peptides. Cambridge University Press.
Cambridge.
Hunter, S.A. and Sanders, J.K.M. (1990). The nature of pi-pi interactions. J Amer Chem Soc 112:
5525–5543.
Wu, G. (2010). Amino acids. Biochemistry and nutrition. CRC Press. Boca Raton, FL.
4 The team

Amides are important substances in the chemical industry, particularly in the phar-
maceutical area. An amide can be formed by a condensation reaction between an
amine and a carboxylic acid. The conventional reaction occurs via an active ester.
Although the reaction appears simple, it is a rather complicated process, requiring
several steps of protecting and deprotecting reactive groups as well as removal of
unwanted by-products (Figure 4.1).

O
R1 COOH + H2N R2 R1 C N R2
H

O
+A* –OA*
R1 C OA* H2N R2

Figure 4.1: Formation of an amide by condensation of a carboxylic acid and an amine.

A nucleophilic attack by the amine on the activated carboxylic acid, in the presence of coupling
reagents and basic solvent, generates a new amide bond. Adapted with permission from
V.R. Pattabiraman and J.W. Bode. (2011) Rethinking the amide bond synthesis. Nature 480:
471–479.

Nature has devised a similar but superior route for the formation of amide bonds or
peptide bonds, as it usually is called in proteins, between amino acids. The conden-
sation reaction occurs on the ribosome, assisted by a template messenger RNA
(mRNA) and a transfer RNA (tRNA) in addition to several other factors (Figure 4.2).
The order amino acids condensate is coded into the mRNA and tRNA carries an acti-
vated amino acid (attached by an ester coupling) to the ribosome. The synthesis is
error free, does not produce unwanted products and can produce very large protein
containing several thousands of amino acid residues. Although it is possible to chem-
ical synthesize proteins (by solid-phase processes), it is not feasible to produce long
(> 100 residues) error-free proteins.

4.1 The peptide bond

The delocalization of the lone pair electrons of the nitrogen to the carbonyl gives the pep-
tide bond ca. 40% double bond character (Figure 4.2B). The bond length between the
peptide bond’s carbonyl carbon and amide nitrogen (1.33 Å) is therefore shorter than a

https://doi.org/10.1515/9783110566185-004
34 4 The team

O O– O O–
C C
+H N Cα H + +H N Cα H
3 3
R1 R2

H2O H2O

H O H O
+H N Cα C N Cα C
3
O– Figure 4.2: Peptide bond formation. (a) Schematic reaction
R1 H R2
mechanism of condensation of two amino acids and the loss
B of a water molecule. The peptide bond can be hydrolyzed by
O O– certain enzyme, proteases. (b) The peptide bond is stabilized
by delocalization of the lone pair of the nitrogen to the
C N C N+
carbonyl, giving the bond between the nitrogen and carbonyl
H H carbon a partial double bond character.

single-carbon nitrogen bond (1.47 Å) but longer than a double-carbon nitrogen bond
(1.27 Å). A planar conformation of the six atoms (carbonyl carbon and oxygen, and
amide nitrogen and hydrogen as well as the two α-carbons) involved in the peptide
group is most stable (Figure 4.3).
The partial double bond character of the peptide has a very important structural
role. There are two possible conformations of the peptide bond; the carbonyl oxygen
and the amide proton can be in a trans or cis conformation (Figures 4.3 and 4.4). As
it turns out, nearly all peptide bonds are in the trans conformation. It is estimated
that only around 0.05% of nonproline peptide bonds are cis, while around 6% of
X-Pro peptide bonds are cis. Due to the double-bond character, there is a rather high
energy cost to change the conformation of a cis peptide bond, and particularly of a
nonproline cis peptide bond, to trans and vice versa.
In contrast to the peptide bond, there is free rotation (within certain limits) of the
bonds between α-carbon and carbonyl carbon and between amide nitrogen and α-
carbon. Since the amide planes are planar, the rotation around each of these bonds
is enough to describe the structure of the chain of amino acid residues or the protein
backbone. For this purpose, two torsion or dihedral angles are defined: Φ (phi) and
ψ (psi). Φ is the rotation of the amide plane around the N–Cα bond and ψ is the rota-
tion of the amide plane around the Cα–C bond (Figure 4.5). The dihedral angles Φ
and ψ are relative to a plane defined by the amide nitrogen, α-carbon and carbonyl
carbon. As the peptide group is planar, the dihedral angle ω is close to +180° (−180°)
or 0° for a trans or cis peptide bond, respectively.
Although a single bond in principle allows free rotation, some conformations
are energetically preferred. A staggered conformation is more stable than an eclipse
 4.1 The peptide bond 35

O
R
1.23 Å H

121.1° 123.2°
N C
1.52 Å 1.33 Å 119.5° Cα

115.6° 1.45 Å
Cα N
119.5° 118.2° C

0.86 Å

H H
R

Figure 4.3: A trans peptide bond. Structural dimensions of average bond lengths and angles are
based on X-ray crystal data from C. Ramakrishnan and G.N. Ramachandran (1965) Stereochemical
criteria for polypeptide and protein chain conformations. II. Allowed conformations for a pair of
peptide units. Biophys. J. 5:909–933, R.A. Engh and R. Huber (1991) Accurate bond and angle
parameters for x-ray protein-structure refinement. Acta Cryst. A 47:392–400 and A. Jabs, M.S.Weiss
and R. Hilgenfeld (1999) Non-proline cis peptide bonds in proteins. J. Mol. Biol. 286:291–304.

O
H
1.24 Å

123° 1.0 Å
121°

119° C N 113°

1.32 Å 1.47 Å
118° 126°
1.53 Å
C
N
Cα Cα

H
H R
R

Figure 4.4: A cis peptide bond. Data from G.N. Ramachandran and C.M. Venkatachalam (1968)
Stereochemical criteria for polypeptides and proteins 4. Standard dimensions for cis-peptide unit
and conformation of cis-polypeptides. Biopolymers 6:1255–1262.
36 4 The team

ω
Φ Cα Ψ ω

Cα N Cα
C

Figure 4.5: Dihedral angles. The dihedral angles Φ, ψ and ω are defined as +180° (or −180°) for a
fully extended chain of amino acid residues.

Staggered Eclipse

Figure 4.6: Newman projection of staggered and eclipse conformations.

conformation (Figure 4.6). The energy difference between a staggered and eclipse
conformation of ethane is ~12 kJ/mol. Thus, there is an energy barrier to free rota-
tion around the single C–C bond in ethane. For any molecule, the most stable con-
formation is the one with the lowest energy, which implies that whenever possible
a molecule would prefer a staggered conformation rather than an eclipse
conformation.

4.2 The Ramachandran graph

When both Φ and ψ are +180° (or −180°), the protein backbone is fully extended and
has a staggered (or staggered-like) conformation. Dihedral angles close to zero give
rise to a forbidden eclipse-like conformation as some atoms get too close to each
other and clashes. Obviously, all dihedral angles that lead to clashes between side
 4.3 The primary structure 37

chains and/or atoms of the backbone are forbidden. Using “normally allowed” and
“outer limit” van der Waals contact distances, Ramachandran and colleagues were
able to define permitted dihedral angles that were supported by analysis of known
structures of di- and tripeptides. The result indicated that all sterically allowed con-
formations fall within two discrete areas, centered around Φ = −60° and ψ = −40°
and around Φ = −120° and ψ = +130° except for dihedral angles involving glycine or
proline. The original conformation space has increased as more and more protein
structures have been solved and the dihedral angles have been determined. The
Ramachandran graphs in Figure 4.7 are based on dihedral angles from structures of a
very large number of actual proteins. The fundamental work by Ramachandran is
still used as a control for determined protein structures, as all dihedral angles must
be within permitted areas; otherwise, the structure probably contains errors. When
validating a protein structure, it is expected that 98% or more of the dihedral angles
fall within the favorable Φ and ψ angles.

A B C
+180 +180 +180
Φ

0 0
Φ

–180 –180 –180

–180 0 +180 –180 0 +180 –180 0 +180
Φ Φ Φ

Figure 4.7: Ramachandran graphs of favorable (dark blue) and allowed (light blue) dihedral angles
from structures of actual proteins. Conformational space of (A) all residues except for glycine and
proline, (B) glycine and (C) proline.
Data from UCSF Chimera. (E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,
E.C. Meng and T.E. Ferrin, T.E. (2004) UCSF Chimera – a visualization system for exploratory
research and analysis. J Comput Chem 25:1605–1612).

4.3 The primary structure

Protein synthesis starts by condensation between the carboxyl group of one amino
acid with the amino group of another amino acid, as shown in Figure 4.2. The re-
sulting molecule now contains two amino acid residues, a dipeptide. The addition
of another amino acid results in a tripeptide and so on until the final polypeptide or
protein is synthesized. This linear sequence of amino acid residues represents the
primary structure of the polypeptide.
The polypeptide has different ends; there is a free amino group at one end and
a carboxyl group at the other end. The end with an amino group is termed the
38 4 The team

N-terminal (or amino terminal), whereas the other end is the C-terminal (or carboxyl
terminal) as shown in Figure 4.8. Therefore, all polypeptides have a direction, from
N- to C-termini.

+ –

N-terminal C-terminal

Figure 4.8: A polypeptide in its fully extended conformation, showing the directionality from the
N-terminal to the C-terminal.

Since the transcription process has the same directionality (from the 5ʹ-end to
3ʹ-end) as the protein synthesis, translation of the template can even begin before
the template is completed.
The backbone of all polypeptides and proteins is identical, a number of amino
acid residues connected by peptide bonds, as shown in Figure 4.8. What distinguish
one polypeptide from another one is the side chains. Thus, the function of any poly-
peptide or protein depends on the properties of the side chains.
Depending on the amino acids composition, the polypeptide may be charged.
In most proteins, the termini as well as other ionizable groups are ionized at neutral
pH. Since the ionization depends on the pH of the solvent, the ionization changes
when pH is altered. At a certain pH, the total number of positive charges equals the
total number of negative charges and the net charge is zero. This particular pH is
called the proteins isoelectric point, pI. At any pH below the pI, the protein will
have a positive net charge and at any pH above the pI it will have a negative net
charge.
As charged groups may be involved in ion–ion interactions that are of great im-
portance for the structure of the protein, altered ionization may affect such interac-
tions, which could have devastating consequences for the protein structure.
Although most proteins contain a few hundred amino acid residues, there are
proteins that only contain a few tens of residues. The largest human protein is the
muscle protein titin that contains around 36,000 residues and has a molecular mass
of 4,000,000 Dalton. Thus, there is an enormous range of sizes among proteins
found in any organism, whether it is of bacterial or human origin.
Before the polypeptide becomes a functional protein, it must be folded cor-
rectly. During the folding process, nonpolar residues are placed away from contact
with the surrounding water and hydrogen bonds and other noncovalent interac-
tions are formed. Since the conformation space is limited by the allowed ranges of
 4.4 The secondary structure 39

Φ and ψ angles, the backbone folds into two major conformations: one helical and
the other extended in addition to loops.

4.4 The secondary structure

The folding process requires not only a driving force to initiate folding but also that the
folded version of the protein is more stable than the unfolded. The driving force of fold-
ing is the hydrophobic effect that leads to minimal contacts between nonpolar groups
and water. During the folding, several noncovalent interactions form: van der Waals
interactions between nonpolar side chains as well as hydrogen bonds and ion–ion in-
teractions. These interactions stabilize the folded conformation of the protein.
In 1951, Linus Pauling and Robert Corey proposed in a series of papers that 3.7
residue α-helices, 5.1 residue γ-helices and pleated-sheets are present in proteins.
They deduced these fundamental structure elements from crystal structures of
small molecules and Pauling’s theoretical framework on chemical bonding. They
also suggested that these structures are stabilized by hydrogen bonds between the
carbonyl oxygen in one residue and the amide hydrogen in another residue. Later
numerous examples of solved protein structures have shown that most of these
predictions were correct, with the exception of the γ-helix that has not been found
in any protein. Today we know that α-helices and β-sheets together with turns
and loops are common structural elements in all proteins. These structural ele-
ments are commonly called secondary structures. When looking at secondary
structures, only the backbone is considered, and the side chains are not involved
in the stabilization of α-helices and β-sheets as well as all other secondary struc-
ture elements.

4.4.1 α-helix

The α-helix is a right-handed coil, with 3.6 amino acid residues per turn or 13 back-
bone atoms. The rise of each turn or the pitch of the α-helix is 5.41 Å. Since there is
no space within the helix, all side chains point to the periphery with a lateral spacing
of 100°. The α-helix is stabilized by hydrogen bonds between the amide C=O group in
residue i and the amide N–H group in residue i + 4, as illustrated in Figure 4.9. The
hydrogen bond length is around 2.8 Å and the dihedral angles Φ and ψ for α-helix
are −57° and – 47°, respectively. An α-helix does not have a specific length but usu-
ally contains between 5 and 40 residues. There are also examples where the helical
length is much longer; the muscle protein myosin is such an example.
The propensity to find a certain amino acid residue in an α-helix varies. A resi-
due with a straight side chain, such as alanine or leucine, occurs more often in an
40 4 The team

Figure 4.9: The α-helix. All side chains are pointing out from the helix. The hydrogen bonds
between the carbonyl oxygens and the amide proton are indicated by the red dotted lines.

α-helix than a residue with a bulky or branched side chain, like tyrosine or valine.
As expected, proline has a very low propensity for α-helices due to its lack of amide
hydrogen and the constrained structure caused by the cyclization. It is perhaps
more surprising that glycine also has a low propensity for α-helices. However, this
is probably because the small side chain (only a hydrogen atom) allows more back-
bone flexibility and the inability to participate in side chain interactions.
A proline residue has no hydrogen atom bound to the amide nitrogen atom due
to cyclization of the side chain. Therefore, the carbonyl oxygen (C=O) of the residue
four residues before the proline has no partner (amide proton) to form a hydrogen
bond with. A proline residue in an α-helical segment is therefore generally consid-
ered as a helix breaker; an α-helix may begin after or end before a proline residue.
This in not entirely correct as there are amino acid sequences with a proline residue
that form noninterrupted α-helices, but the presence of the proline creates a kink in
the helix instead of ending the helix (Figure 4.10).
As the peptide bond has a trans conformation, the amide N–H and C=O groups
in the same peptide unit are pointing in opposite directions. As both the amide hy-
drogen and the carbonyl oxygen have partial but opposite charges, the peptide unit
is polarized and has a weak dipole moment. At the N-termini, the amide N–H groups
in the four most N-terminal residues have no amide C=O group to form hydrogen
bonds with. Analogous at the C-terminal, the amide C=O groups in the four most C-
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Title: Toinen tai toinen naimaan: Ilveily yhdessä näytöksessä

Author: A. Wilhelmi

Translator: S. K. Hämäläinen

Release date: May 31, 2016 [eBook #52198]

Language: Finnish

Credits: Produced by Tapio Riikonen

*** START OF THE PROJECT GUTENBERG EBOOK TOINEN TAI

TOINEN NAIMAAN: ILVEILY YHDESSÄ NÄYTÖKSESSÄ ***
Produced by Tapio Riikonen

TOINEN TAI TOINEN

NAIMAAN
Ilveilys yhdessä näytöksessä

Kirj.

Alexander Victor Wilhelmi

Suomennos [S. K. Hämäläinen]

Näytelmäkirjallisuutta N:o 8.
Kuopiossa, U. W. Telén & Co, 1897.
HENKILÖT:

Jaakko Grimm. | Veljeksiä. Yliopiston professoreja.

Wilhelmi Grimm. |
Gertrud. Heidän tätinsä.
Loviisa. Gertrudin veljen tytär.

(Näyttämö: puutarha, oikealla asuinhuoneesen vievä ovi, samalla

puolella myös huvihuone tai avonainen lehtimaja; huvihuoneessa
pöytä, peitetty kirjoilla, pallokartoilla ja fysikaalisilla koneilla; toisella
puolella puun juurella pöytä ja tuoleja; perällä huvihuoneen puolella
on pensas.)

Ensimmäinen kohtaus.

Jaakko ja Wilhelmi (istuvat huvihuoneessa mukavilla

nojatuoleilla, vaipuneina lukemiseen). Rouva Gertrud (vähän
ajan kuluttua tulee huoneesta).

Gertrud. No niin! Tuossa nuo kirjatoukat taas nuhjustelevat nenä

kiinni vasikan nahassa. Ei ne ota tietääkseenkään, vaikka maailma
menisi nurin, narin, ei vaikka maa halkeaisi!

Hä! te kirjatoukat, te nahkamadot! aamiainen odottaa jo aikaa

sitte!
Vai ilmastako te elätte?

Wilhelmi (nostaa päätänsä rauhallisesti). Johan te olette sen meille

kolmasti kertonut.

Jaakko (samoin). Täti kulta, älkää häiritkö meitä! Ei hätää

pahaakaan!

(Kumpikin jatkaa lukuaan.)

Gertrud (vihasesti). Vai niin? Luuletteko te ettei minulla ole muuta

työtä kuin odottaa, milloin te kahville käytte? Kaiken päivääkö sitä
pitää varistaa? (Wilhelmille.) Ja juuri koska sen olen jo kolmasti
kertonut, pitäisi teidän totteleman.

Wilhelmi (hänestä huolimatta Jaakolle). Bulgarien syntyperä on

aivan varmaan Hindostanissa. Heidän ja Petcheneegien kielen
yhtäläisyys osoittaa selvään —

Gertrud (vimmastuneena). Viis bulvaaneille, kalmukeille ja

koirankuonolaisille; tämä on liikaa! Ikäänkuin meidän kalttaisia
ihmisiä ei maailmassa olisikaan olemassa!

Jaakko. Huutonne, täti kulta, osoittaa selvään, että kyllä te ainakin

olette olemassa.

Wilhelmi. Aivan hyvä olisi, jos ette joutavilla häiritsisi

tutkimisiamme.
 Gertrud. Joutavillako jutuilla? Oiva aamiainen, kahvi, munat, paisti;
ovatko ne joutavia? Ainoastaan teidän kalttaisenne läkkisormet
saattavat niin sanoa. Sanokaapas mikä teidän tieteellisistä
tuhmuuksistanne olisi tärkeämpi!

Jaakko. Täti kulta, te ette sitä käsitä.

Gertrud (pikaistuen). No kaikkea vielä! Pitäisikö minunkin

tuommoisilla loruilla päätäni pyörryttää? Kyllä minä tiedän miksi te
minua noin halveksien katselette; vaan kumminkin te ilman minutta
saisitte ripustaa hampaanne naulaan.

Wilhelmi (pikaisesti). Kyllä kai! Me pitäisimme teidän

kelvollisuutenne aivan hyvässä arvossa, jos te vaan ette aina sillä
niin kerskusi.

Gertrud. Katsopas vaan! Johan Jobikin tästä tuskastuisi. Se on

synti ja häpeä, että kaksi nuorta, oivallista ja rotevaa miestä näin
kuivettuu kirjainsa toimissa, kuin savustetut sillit uunin piipussa.

Wilhelmi. Mikä proosallinen vertaus!

Gertrud. Mutta totinen ja tuntuva! Tuuman paksuinen pöly peittäisi

teidätkin, niinkuin kirjanne kannet, joll'en teitä välistä karistelisi. Ja
mitä te sitten oikeastaan tiedätte? Ette mitään — ette rahtuakaan!

Jaakko. Aivan oikein, valitettavasti liian oikein! Ihmisen tieto on

vaillinainen.

Gertrud. Niin, mokomien narrien tieto. Muut ihmiset, jotka eivät

näin koko päivää istu nenä kirjassa, tietävät kyllä mikä on kohtaus ja
oikeus. Ne osaavat hoitaa taloaan ja omaisuuttaan, osaavat elää niin
että sekä itsellään että muilla on siitä iloa.
 Jaakko. Tieteet olisivat vielä huonommalla kannalla kuin ovat, jos
kaikki noin ajattelisivat.

Wilhelmi. Oppimaton maailma ei voi pitää oppineita liian korkeassa

arvossa. Rahan kokoaminen, syöminen, juominen ja makaaminen,
nekö mielestänne ovat elämä? Useat eivät tiedäkään elävänsä ja
miksi he maailmaan ovat luodut. Täti, miksi te oikeastaan elätte
maailmassa?

Gertrud (vähän hämillään). Mitä? Minäkö? Miksikö elän

maailmassa? Onpas kaikkea! Minä, minä elän huvikseni.

Wilhelmi. Sekin muka syy! Ei kelpaa, sanokaa parempia.

Gertrud (vihasesti). Miksikä elän? Sitä en ole tullut tuumanneeksi.

Sitä ei minulle ole kukaan sanonut. Minä elän sitä varten, että
tuommoiset puustavit ihmishaamussa saisivat minua kiusata ja
äköittää. Sitä varten, että te saisitte kaiken huoleni ja vaivani palkita
kiittämättömyydellä, siksi että te saisitte toivottaa kuolemaa tälle
vanhalle tädille, joka on teitä ruokkinut, vaatettanut ja pitänyt
murheen teidän vähäisestä taloudestanne. Mielestänne varmaan
olen liian sitkeähenkinen. Minä tiedän jo olevani teille kuormaksi!

Wilhelmi. Vaan eihän siitä ole puhettakaan, täti kulta!

Jaakko (Wilhelmille). Kuka mitä kaapii, se sitä saapi. Mitä rupesit

kinaan vaimoväen kanssa. Eihän ne pysy koskaan asiassa.

Gertrud (vimmastuen Jaakolle). Vai niin! Siis meidän kanssa ei

maksa edes puhellakaan! Aivan oikein, sinä olet vanhempi ja osoitat
siis hyvää esimerkkiä. Neuvollasi turmelet vielä hänet kokonaan.
Hänellä toki vielä on hiukka sydäntä ja tunnetta. Mutta sinä! Paras
olisi jos nidottaisit itses parkittuun pergamenttiin ja asettaisit itses
kirjakaappiin joutavien lorukirjasi sekaan.

Wilhelmi. Siinä sait! Se oli aivan oikein sinulle! Kuka käski sinun
sekaantua meidän riitaamme?

Gertrud (kävellen edestakaisin). Tämä ei käy laatuun; muutos on

tehtävä ja vielä tänä päivänä! Kylläpä te saatte kyytinne! (Lähenee
heitä vakaisena.) Teidän pitää naida! Pari rouvaa pitää tulla taloon!
Sitten täytyy teidän ihmistapoihin oppia.

Wilhelmi | (hämmästyen). Taivaan herra! Jaakko |

Jaakko. Herran tähden, täti, älkää taas uudistako vanhaa

uhkaustanne!

Wilhelmi. Tule veli, tehkäämme hänelle mieliksi ja mennään

syömään!

Gertrud. Ähä! Jopa sattui! (ääneen.) Niin, naida teidän täytyy ja

tällä kertaa on se täyttä totta. Minä olen kyllin teille ehdotellut
neitosia, valitkaa nyt!

Jaakko. Vieläkö meidän pitää teille taas kertoa, että siinä asiassa
on tarkoin tuumailtava.

Gertrud. Ikäsikö sinä tuumailisit? Katsopas peiliin ja ole iloinen, jos

joku neito huolisi tuommoisesta harakan hirmusta.

Wilhelmi. Naimiseen on meillä vielä kyllin aikaa. Ei kiirettä

kirkkoon, ei hätää häihin! Millä oikeudella te meitä noin kiiruhdatte?
 Gertrud. Milläkö oikeudella? Kuka on teidät kasvattanut? Kuka
käsillään kantanut? Kuka opettanut seisomaan ja puhumaan? Tosin
niin kauas ei teidän muistinne riitä, mutta minä, minä joka olen
rakastanut teitä kuin omia lapsiani, minä en sitä ole unhottanut.

Jaakko. Koska te siis meitä rakastatte, niin älkää häiritkö

onneamme; sallikaa meidän rauhassa ja hiljaisuudessa jatkaa
oppiamme ja tutkimisiamme.

Gertrud. Minä tiedän paremmin, mitä teidän rauhaanne sopii.

Joll'ette tottele minua ja heti päätä, erkanen minä teistä ja teen
teidät perinnöttömiksi.

Wilhelmi. Hyvä täti, emmehän me haluakaan teidän

omaisuuttanne.

Jaakko. Vaatimuksemme ovat vähäiset ja tarpeemme tuiki pienet.

Me mielellämme luovumme rahoistanne ja tavaroistanne, kunhan
vaan ette kiusaa meitä naimisiin.

Gertrud. Hyvä, olkoon niin! Mutta lähden samassa ja jätän teidät

onnenne nojaan. Saadaanpa nähdä, etteköhän ole kuin kissa
liukkaalla jäällä.

Jaakko | (hämmästyneinä). Täti kulta, mitä te ai'otte?

Wilhelmi |

Gertrud. Minä jätän tämän talon, sillä tämmöistä elämää en saata

kärsiä. Vaikka muka olette niin suuresti oppineet, olette te kuitenkin
sitten pahemmassa kuin pulassa, sillä kaikista noista suurista
kirjoistanne ette saa tietää, kuinka hoidetaan taloa, vaatteita,
kyökkiä ja kellaria. Sitte vasta opitte pitämään arvossa vaimon käsiä,
vaikka ne olisivatkin vaan vanhan tädin kädet.

Jaakko | (hämillään katselevat tosiaan). Herran Wilhelmi |

tähden, täti, älkää toki noin peljättäkö!

Gertrud. Hävetkää toki! Te ette koskaan tee mitään vanhan tätinne

mieliksi. Ja joll'ette minusta pidäkään väliä, pitäisihän teidän toki
muistaa kuolevan isänne tahtoa. Minä tosin en ole kouluja käynyt,
vaan kuitenkin tiedän vanhempain tahdon täyttämisen olevan pyhä
velvollisuus.

Wilhelmi (masentuneena Jaakolle). Se on totta, se oli isämmekin

viimeinen tahto.

Jaakko. Se oli hänen viimeinen sanansa (tädille). Vaan täti, hän

sanoi, että vaan toinen meistä välttämättömästi piti mennä naimisiin.

Wilhelmi (rohkeammin). Vaan te vaaditte kumpaakin naimisiin.

Mitähän me kahdella rouvalla tekisimme. Silloinhan ei olisi mitään
rauhaa talossa.

Gertrud, Sitä et sinä käsitä! Mitä enemmän rouvia, sitä parempi

osa. Kunhan vaan toinen alkaa, kyllä toinen seuraa. Mutta toisen
teistä nyt täytyy alkaa.

Jaakko (kaapien korvaansa, veljelleen). Siis toisen meistä täytyy

naida. —

Wilhelmi. Eihän tässä muu auttane!

Jaakko. Noh, veli! Mitä mietit? Sinä olet vielä nuori; sinä saatat
koettaa.
 Wilhelmi (peljästyen). Jumala varjelkoon! Sinä olet vanhempi,
sinun on alettava.

Jaakko. Sinä pikemmin sovit siihen. Sinulla on somemmat tavat ja

temput ja suoraan sanoen oletpa myös kauniinlainen.

Wilhelmi. Sinä olet paljon vakavampi ja miehekkäämpi; sinä olet

paljon sopivampi aviomieheksi kuin minä.

Jaakko. Minä en voi, se on mahdotonta minulle!

Wilhelmi (epätoivossa). Minulle myös! Minä en taida!

Gertrud (suuttuneena keskeyttäen). Taasko sitä vanhaa häilymistä

ja horjumista? Siinä nyt seisotte kuin pitäisi hirteen mennä.
Rohkaiskaa toki sydäntänne, te jänikset, eihän se teiltä henkeä
hukkaa!

Jaakko. Wilhelmi oli aina rohkeampi minua.

Wilhelmi. Jaakon olisi pitänyt jo aikoja sitten alkaa. Hän ei ole

huolinut yhdestäkään neidistä, vaikka te olette sen tuhannetkin
hänelle esitelleet. Valitkoon nyt yhden niistä!

Jaakko. Ne ovat varmaan jo kaikki kuolleet, taikka menneet

naimisiin. (Tädille.) Minä olen varma siitä, ettei teilläkään tällä
haavaa ole ketään esitettävänä.

Gertrud. Eikö vaan olisi? Vaikkapa kymmenen! Ja onpa aika kelpo

tyttö niiden joukossa, liiankin hyvä teille. Mutta te nahjukset ette
huomaa, vaikka teitä nenästä vetäisi. Ei teillä ole silmiä eikä
kauneuden tunnetta!
 Wilhelmi. Kuinka niin, täti?

Gertrud. Ettekö siis ole ollenkaan huomanneet, miksi tuotin

luokseni velivainajani tyttären?

Jaakko. Loviisanko?

Gertrud. Hän ei ole rikas, mutta kaunis ja oiva neiti. Siis pitkittä
puheitta päättäkää nyt heti, sillä ei kaikki kypsy, jota kauan
keitetään. Toisen teistä täytyy tohvelia totella. Miettikää siksi, kunnes
palaan, vaan sitte tahdon tietää, kumpiko teistä on tuleva sulhoksi.

(Menee huoneesen.)

Toinen kohtaus.

Jaakko ja Wilhelmi (kävelevät miettivinä, kädet selän takana).

Jaakko (hetken kuluttua). Saakelin kohta!

Wilhelmi. Kirottu seikka!

Jaakko (aina vaan kävellen). Pääni on kuin pyörällä. Mitenkähän

sitten käynee, kun rouva taloon tulee!

Wilhelmi (samoin). Kauheata! Hirveätä! Eiköhän mitään keinoa

pelastukseen! Minkä näköinen se serkku sitten lienee?

Jaakko. En ole vielä häntä edes silmäillytkään.

Wilhelmi. En minäkään. Mutta täti ei anna meille rauhaa, kyllä

minä sen tiedän. Hän täyttää uhkauksensa ja jättää meidät.
 Jaakko. Mitä me sitte teemme? Me olemme niin perin juurtuneet
vanhoihin tapoihimme.

Wilhelmi. Niin kyllä! Rohkase mielesi ja ota rouva!

Jaakko. Minäkö? En ikänäni. (Tarttuu veljensä käteen, hellästi.)

Veliseni, ole järkevä, sinä olet nuori, täynnä toivoja, täytä pyyntöni,
nai sinä serkkumme!

Wilhelmi (samoin). Veli kulta, kaikki mitä tahdot, vaan en tätä!

Jaakko. Katsoppas, minä tiedän, että sinä ennenkin olet naisia

miellyttänyt. Sinua on myöskin jo ennen rakastettu. Minulle ei
koskaan ole semmoista sattunut; minä olisin aivan kuin tanssiva
karhu.

Wilhelmi. Eipä ne olekaan sen hullumpia. Kerran näin semmoisen

elävän — — —

Jaakko. Sinulla on enemmän taipumusta naimiseen. Sinä tulisit

varmaan onnelliseksi. Avioliittoa ja perhe-elämää kehuvat niin
viehättäväksi. Minä jo kuvittelen itselleni sinua ihanan, oivallisen
vaimon puolisoksi, viehättävien ja onnellisten lasten isäksi; voi
kuinka ne sinua halaavat ja suutelevat, sinä hypität niitä polvillasi, ja
te kaikki elätte rakkaudessa ja onnessa. Usko minua, semmoista
onnea ei jokainen saavuta!

Wilhelmi. Noh, hyvä veli, hanki itsellesi tuo onni!

Jaakko. Eihän minusta ole puhettakaan. Minulla ei ole taipumusta

avioliittoon, vaan sitä enemmän minä iloitsen sinun onnestasi. Minä
kasvatan lapsiasi ja rakastan niitä kuin omiani. Ja ajattelepas, kuinka
kaunis tyttönen Loviisa on!
 Wilhelmi. Ethän häntä ole vielä nähnytkään.

Jaakko. Noh, minusta vaan niin tuntui ja niinhän tätikin kertoi.

Päälle päätteeksi on hän niin hyvä ja oiva neiti. Et varmaankaan
kadu kauppaasi.

Wilhelmi. Niinhän se tätikin sanoo. Siis toivotan vaan sinulle

onnea!

Jaakko (tuskaisena). Taivaan Herra, eihän tuohon pysty mikään!

Minä koetan kaikki voimani, kuvaan avioelämän mitä
viehättävimmillä väreillä ja sinä pysyt tunteettomana kuin kivi.
Sanohan taivaan tähden, miksi siis et tahdo mennä naimisiin?

Wilhelmi. Miksi ei sinua haluta?

Jaakko. Etkö ymmärrä etten minä saata?

Wilhelmi. Etkö ymmärrä etten minäkään saata?

Jaakko. Siis sinä varmaankaan et tahdo.

Wilhelmi (kylmäkiskoisesti). Minä en voi.

Jaakko. Nyt tiedän minkä verran sinä veljeäsi rakastat.

Wilhelmi. Jos sinä minua rakastaisit, niin sinä itse menisit

naimisiin. Vaan osoittaakseni etten ole niin kovasydämminen kuin
sinä, esittelen seuraavan keinon.

Jaakko. Minkä?

Wilhelmi (päättävällä muodolla). Heittäkäämme arpaa!

 Jaakko. Arpaako? Ei, se olisi kevytmielistä.

Wilhelmi. Niin minustakin; senpätähden nai suorastaan.

Jaakko. Saattaahan arpa kohdata minuakin.

Wilhelmi. Tosin, vaan paha kyllä myös minuakin.

Jaakko. Mitä minä sitten teen?

Wilhelmi. Menet naimisiin.

Jaakko. En minä rupea arpaa heittämään; en minä koskaan tahdo

antaa itseäni semmoisen vaaran alaiseksi.

Wilhelmi. Olkoon niin! Mutta silloin sinun varmaankin täytyy naida,

sillä minä en sitä tee.

Jaakko (rukoillen). Veliseni!

Wilhelmi (äkäisesti keskeyttäen). Jätä minut jo rauhaan!

Jommankumman se täytyy tehdä. Kumpikaan ei tahdo, siis arpa
määrätköön, kumpi on uhriksi joutuva. Tämä on viimeinen sanani ja
ainoa mitä sinulle mieliksi saatan tehdä.

Jaakko. Noh olkoon sitten! Vaan kuinkas tässä menetellään?

Wilhelmi. Siinä ei suurta taitoa tarvita. (Menee pöydän ääreen.)

Me valitsemme kaksi palloa, toisen valkean, toisen mustan.

Jaakko (tuskallisesti kertoen). Toisen valkean, toisen mustan.

Wilhelmi. Täällä ei nyt ole niitä yhtään.

 Jaakko (iloisena). Jumalan kiitos!

Wilhelmi. Sitä parempi, me otamme kaksi lappua.

Jaakko (kertoen). Kaksi lappua.

Wilhelmi. Näin, toiseen piirustan ristin, toinen jääpi puhtaaksi.

Jaakko. Toiseen ristin.

Wilhelmi. Risti velvoittaa avioliittoon. (Käärii sen.)

Jaakko. Tämä on oikein asian laidan mukaan.

Wilhelmi. Puhdas lappu vapauttaa naimisesta.

(Käärii senkin.)

Jaakko. Voi, jos sen saisin!

Wilhelmi. Kyllä kai; mikä hätä sitten olisi arvan heittoon ruvetessa!

Jaakko. Mitäs sitten tehdään?

Wilhelmi (hakien). Nyt tarvitsemme uurnaa; paremman puutteessa

otan sinun yölakkisi (tavoittaa Jaakon päätä).

Jaakko. Älä, veli veikkoseni! Ota omasi, minulla on paha onni!

Wilhelmi. Tahtosi tapahtukoon (ottaa omansa, heittää laput siihen

ja puistelee lakkia). Näin nyt onnemme seulotaan!

Jaakko (vavisten). Mutta rehellisesti, veli, rehellisesti.

Wilhelmi. Arvattavasti! Oikeus ja kohtuus! Noh, ota nyt!

 Jaakko. En — minä — en uskalla — ota sinä!

Wilhelmi. Älä tee verukkeita, ota joutuisaan!

Jaakko. Totisesti, minä en uskalla. Minä varmaan saan tuon

onnettoman ristin. Ole hyvä, ota ensin!

Wilhelmi. Vai sekin vielä! (Puistelee kauan lakkia, ottaa lapun ja

ojentaa lakin Jaakolle; kylmäkiskoisesti.) Ota!

Jaakko (tuskallisesti lähenee lakkia, vapisevana ja huokaillen ottaa

lapun). Näin!

Wilhelmi (heittää lakin pöydälle). Nyt on kummallakin osansa.

(Kurottaa lappuaan.)

Jaakko (samoin). Niin, vaan minä vapisen kuin haavan lehti!

Wilhelmi. Auaistaan vaan viipymättä!

Jaakko. Minä en uskalla; aukaise sinä ensin!

Wilhelmi (äkäisesti). Miksikä minun aina pitää kaikki ensin

tekemän? Ei nyt auta, avatkaamme molemmat samalla kertaa! Minä
luen kolmeen asti. (Lukeissaan käypi verkalleen pöytää kohden;
Jaakko puuta kohden.) Siis yksi!

Jaakko (läheten puuta). Yksi!

Wilhelmi (edelleen käyden). Kaksi!

Jaakko (samoin, vapisee). Kaksi!

 Wilhelmi. Kolme! (aukaisee).

Jaakko (on au'aissut lappunsa ja huomannut ristin). Voi, nyt tuho

tuli!

Wilhelmi. Hei, hurrah! Nyt iloitaan!

Jaakko (vaipuen tuolille). Voi kovaa onnea, voi onnettomuutta!

Wilhelmi (iloiten). Nyt olen vapaa, vapaa kuin lintu, vaimojen

pauloista irti jo päässyt! Hei, hurrah! Tahtoisinpa syleillä koko
maailmaa!

(Hyppää ympäri.)

Kolmas kohtaus.

Edelliset. Gertrud (tulee huoneesta).

Gertrud (ihmetellen). Mikä meteli täällä on? Wilhelmi, oletko

pyöräpäinen?

Wilhelmi (syleillen ja tanssittaen häntä). Saanko syleillä, saanko

suudella? Minä olen onnellisin auringon alla!

Gertrud. Päästätkö irti, sinä tuulen tuoma! Kymmeneen vuoteen

en ole sinua tuommoisena nähnyt, mitä on tapahtunut? (Huomaa
Jaakon.) Ja mikäs tuota vaivaa? Toinen hyppii ja tanssii kuin hullu,
toinen on kuin ukkosen lyömä.

Wilhelmi (tragikoomillisesti osoittaen Jaakkoa). Ei, täti, hän on

vaan kamppauksen kestänyt ja päätöksen tehnyt; se on häntä niin
väsyttänyt.
 Gertrud (iloisesti). Noh, Jaakko!

Wilhelmi. Niin täti, hän päätti mennä naimisiin.

Gertrud (taputtaa Jaakkoa olalle). Kas niin, se oli miehen työ!

Viimeinkin viisastuit!

Jaakko (tointuu ja nousee ylös). Voi minua kovan onnen

leikkikerää! Kyllähän minä sen kohta arvasinkin! Minunko pitää
naimisiin!

Gertrud (tyytyväisenä hieroen käsiään). Siis Jaakko on tuo

onnellinen! Sepä on sangen hauskaa! Tiesinhän minä että hän on
järkevämpi. Seuraa sinä nyt veljeäsi!

Wilhelmi. Te olette oikeassa, täti. Jaakko on hyväsydämminen

poika. Me tuumailimme tyyskästi asiaa ja hän suostui
vapaaehtoisesti täyttämään tahtonne.

Jaakko (toiselta puolelta läheten Wilhelmiä; hiljaan). Vaan minäpä

en sitä tee. Asia ei ole käynyt rehellisesti. Sinä otit ensin ja siten sinä
voitit.

Wilhelmi (pikaistuen). Ethän sinä rohjennut ensin ottaa. Älähän

toki enään revi asiaa auki uudelleen.

Jaakko. Täti sen ratkaiskoon!

Wilhelmi (hiljaa ja vakuuttavasti). Ethän toki hänelle ilmoittane

meidän heittäneen arpaa. Sehän olisi aivan sopimatonta.

Jaakko. Olisiko niin?

 Wilhelmi. Aivan varmaan! Sinä häpäisisit itsesi ijäksi päiväksi. Ole
nyt järkevä ja tyydy kohtaloosi!

Gertrud. Mitä juonia te siellä taas kudotte?

Wilhelmi. Ei mitään erinomaista; Jaakko vaan ei tiedä miten

varustaisi häätoimet, puvut ynnä muut tarpeet.

Jaakko. Puvutko ja tarpeet?

Gertrud. Ohoh! Älkää siitä surko, ne minä paraiten toimitan! Sen

toimen tunnen minä perinpohjin ja jo tänään ryhdyn työhön.

Jaakko. Eihän tässä vielä kiirettä ole.

Gertrud. Onpa maar kiirettä! Pitää siivota huoneet, varustaa

vaatteet, laittaa ruo'at. Tarvitaanpa vielä uusia huonekaluja,
kyökkitarpeita, morsiusvaatteita ja tuhansia muita tarpeita.
(Tyytyväisenä.) Niin, niin Jaakko, luota vaan tätiisi. Minä en unhota
mitään, en edes lapsen sukkia ja paitojakaan.

Jaakko (pannen kädet ristiin). Taivas! Lapsen paidatkin!

Wilhelmi (ilkkuen). Sukat myöskin.

Gertrud. Lapseni, minä olen ikäänkuin kymmenen vuotta

nuoremmaksi tullut; Jumala sinun päätöstäs siunatkoon, Jaakko!
Pane vaan päätöksesi heti toimeen, sillä parempi pyy pivossa kuin
kaksi kuusessa.

Jaakko (pyyhkien hikeä otsastaan). Kyllä nyt hikoillaan.

Wilhelmi (katsellen vasemmalle). Ellen erehdy, tulee Loviisa

par'aikaa puutarhasta.
 Gertrud (katsoen). Oivallista, hän tulee kuin kutsuttu! Siis Jaakko
asiaan käsiksi; kosi nyt heti!

Jaakko. Nytkö heti?

Gertrud. Se on tietty; ja ole oikein hempeä ja hieno, ettet saisi

rukkasia.

Jaakko. Kunhan ne vaan saisin!

Gertrud. Vaan minkä näköinen sinä olet! Kampaa tukkasi ja

partasi! Heitä nurkkaan tuo yönuttusi ja pukeudu uuteen mustaan
takkiin taikka vielä paremmin frakkiin.

Jaakko. Ei minulla taida frakkia ollakaan.

Gertrud. Entäs se uusi, joka oli päälläsi maisteritutkinnossa?

Wilhelmi. Kunhan vaan koit eivät olisi sitä syöneet. Tule, Jaakko,
minä puen sinut oikein prinssiksi!

Jaakko. Sano paremmin alttarille vietäväksi uhriraavaaksi. (Tätille.)

Vaan saattepa nähdä, täti, että minä saan rukkaset.

Wilhelmi (taluttaen häntä pois). Vielä mitä, noin pulska poika,

koristettu ja kaunistettu, puettu mustaan frakkiin ja valkoiseen
liinaan, saapi vaikka kymmenen joka sormelle, ja ettei Loviisa sinulle
rukkasia anna, se olkoon minun huolenani.

(Viepi hänet huoneesen.)

Gertrud (juosten jälestä). Menkää, menkää! Kohta on Loviisa

täällä. Laittaukaatte pian valmiiksi!
 Neljäs kohtaus.

Gertrud (yksin) sitten oitis Loviisa.

Gertrud (palaten). No viimeinkin ollaan näin pitkällä — mutta

kylläpä siinä saikin vaivaa nähdä! Nyt pitää vaan kuulustella Loviisaa.
Olisinpa aika pulassa, jos hän puolestaan nyt panisi vastaan.

Loviisa (vasemmalta, lukien, kirjaa).

Gertrud. Mutta mitä minä tuossa näen! Kirja kädessä! Tuota vielä
puuttui! Tarttuuko tuo rutto häneenkin! Loviisa, mitä tämä on? Mitä
sinä lueskelet?

Loviisa. Täti kulta, tämä on oiva kirja! Se on Wilhelmin uusin teos,

hänen matkakertomuksensa Pohjoismaista. Voi kuinka nerollista,
kuinka kaunista! Sitä lukeissaan aivan luulee näkevänsä nuo seudut
ja ihmiset. Kuinka tarkka tapojen ja luontojen kuvaus, kuinka syvät
mietteet, kuinka sopivat arvostelut! Ah, Wilhelmi se on nerollinen
mies!

Gertrud. Vai niin! Wilhelmikö? Jätähän nuo joutavat, ne panevat

vaan pääsi pyörälle, eivätkä hyödytä rahtuakaan.

Loviisa. Mikä sitten olisi hyödyllisempää kuin hyvä kirja, erittäin

näin opettavainen kuin tämä Wilhelmin teos.

Gertrud. Heitä jo Wilhelmisi! Jaakko kirjoittaa myös kirjoja ja

vieläpä paksumpiakin.

Loviisa. Saattaa olla, vaan ne ovat kreikan tai latinankielisiä; minä

en niitä ymmärrä. Vaan Wilhelmin kirjat — — —
 Gertrud (pikastuen). Jätä toki jo tuo Wilhelmi sikseen! Nyt on
muuta mietittävää. Sano, miltä tuntuu olosi täällä?

Loviisa. Kyllä vallan hyvältä; välistä kuitenkin on vähän yksinäistä.

Gertrud. Miellyttävätkö serkkusi sinua?

Loviisa. No kyllä ne ovat hyvin totisia ja sangen harvapuheisia.

Jaakko ei ole minua edes nähnytkään. Luullakseni hän on
jörömäinen. Sitä vastoin Wilhelmi — —

Gertrud. Niin kai, kyllä, kai. Mutta Jaakko on pohjaltaan hyvä ja

kunnon mies, sen sanon sulle.

Loviisa. Sen kyllä uskon; kuitenkin täytyy olla jotenkin rohkea,

ennenkuin häntä tohtii katsellakaan. Minusta tuntuu aina ikäänkuin
hän olisi minulle vihoissaan. Wilhelmin katsanto on paljon leppeämpi.

Gertrud (äkäisesti). Vai niin! Wilhelmikö on leppeämpi. (Astuen

sivulle, vihoissaan.) Kas niin! Tääpä on kaunis juttu, kun yksi este on
poissa, on toinen jo edessä. Nyt on taas tyttönen mieltynyt
Wilhelmiin. Aivanhan tässä pakahtuu sulasta harmista, kun työ ja
vaiva näin käy turhaksi!

Loviisa (ihmetellen). Tätiseni, mikä teitä vaivaa?

Gertrud. Mutta hän ei saa saattaa hankettani hukkaan; kyllä minä

avaan silmät häneltä. (Loviisalle.) Sinä et ole oikein tarkastanut
Jaakkoa. Hän on siveä ja lempeä mies. Katsoppas vaan hänen
kauniita, sinisiä silmiään. Ne ovat paljon leppeämmät ja suloisemmat
kuin Wilhelmin.

(Jatkaa puhettaan Loviisan kanssa.)

 Viides kohtaus.

Edelliset. Wilhelmi, Jaakko (pitkässä suippoliepeisessä frakissa

ja valkoisessa liinassa).

Wilhelmi (tuopi veljeään huoneesta). Kas niin, rohkeasti eteenpäin

vaan! Tosiaan, sinä olet oikein soman näköinen.

Jaakko. Wilhelmi, minä joudun koko maailman nauruksi.

(Pysähtyvät oikealle ja puhelevat keskenään.)

Gertrud (huomaten heidät). Tuossa hän onkin. Katso toki kuinka

kaunis! Komea vartalo, jalo muoto!

Jaakko (Wilhelmille.) Luullakseni täti jo häntä neuvottelee.

Gertrud. Elä ole noin kaino! Katso suoraan; kyllä hän sitten on
ystävällisempi. (Jaakolle lähtiessään.) Minä jätän teidät nyt kahden
kesken. Wilhelmi tulkoon kanssani. Aja nyt hyvin asiasi. —

Jaakko. Ei, Wilhelmi jääköön tänne; yksinäni en saa mitään

toimeen.

(Katselee pelolla Loviisaa.)

Gertrud. Niinkuin tahdot. (Menee Loviisan luo, joka on istunut

pöydän ääreen.) Huomaatko kuinka leppeästi hän sinua katselee?
Kun hän sinua puhuttelee, niin ole sinäkin puoleltasi lempeä ja
hempeä hänelle; ymmärrätkö? (Menee taas Jaakon luo.) Noh,
Jaakko, eteenpäin, rohkase sydämmes! Tuommoiseltako kosiomies
näyttää? Jos vaan minä olisin mies, en minä noin kuhnustelisi;
tuulessa ja tuiskussa minä tytöt veisin. Sinä jänisjalka!
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