Proteins: Their Primary, Secondary, Tert

iary, and Quaternary Structures

蛋白質:初級、次級、三級及四級結構

中國醫藥大學 醫學系黃琮竣 副教授

 本章節之學習目標
有哪些非共價鍵結作用穩定蛋白質結構？

次級結構有哪些形式及其形成方式？

多肽鏈如何折疊成三級結構？

蛋白質次單元之交互作用在蛋白質的四級結構中所扮演的角
色。
 5.1 What Architectural Arrangements Characterize
Protein Structure?

• Proteins are classed according to shape and solubility:

• Fibrous proteins（纖維蛋白） have relatively simple,
regular, linear structures
• Globular proteins（球蛋白） are roughly spherical in
shape
• Membrane proteins（膜蛋白） are found in
association with the various membranes of cells
5.1 What Architectural Arrangements Characterize
Protein Structure?

Figure 5.1 (a) Proteins having structural roles in cells are typically
fibrous and often water insoluble. (b) Myoglobin is a globular
protein. (c) Membrane proteins fold so that hydrophobic amino
acid side chains are exposed in their membrane-associated
regions.
 Protein Structure is Described in Terms of
Four Levels of Organization

• The four levels of protein structure are:

- Primary (1°) – the amino acid sequence
- Secondary (2°) - local structures stabilized
by H bonds
- Tertiary (3°) - overall 3-dimensional shape
of the folded protein
- Quaternary (4°) - subunit organization
 5.1 What Architectural Arrangements
Characterize Protein Structure?

Figure 5.2 Bovine

pancreatic ribonuclease A
contains 124 amino acid
residues, none of which
are Trp. Its four disulfide
bridges are indicated in
gold.
 5.1 What Architectural Arrangements Characterize
Protein Structure?

Secondary structures
in proteins

Figure 5.3 The α-helix and

the β-pleated strand are the
two principal secondary
structures found in proteins.
 How to view a protein?

• The tertiary structure of a protein may be viewed in

several ways (Figure 5.4):
• Backbone only

• Backbone plus side chains

• Ribbon structure

• Space-filling structure

• Each of these is an abstraction

How to view a protein?
The Quaternary Level of Protein Structure

Figure 5.5
Hemoglobin is a
tetramer consisting
of two α and two β
polypeptide chains.
Computer Programs Can Align Sequences
and Discover Homology Between Proteins

Alignment of the amino acid sequences of two protein homologs

using gaps. Shown are parts of the amino acid sequences of the
catalytic subunits from the major ATP-synthesizing enzyme (ATP
synthase) in a representative archaea and a bacterium. These
protein segments encompass the nucleotide-binding site of these
enzymes.

Identical residues in the two sequences are shown in red.

Introduction of a three-residue-long gap in the archaeal sequence
optimizes the alignment of the two sequences.
 5.7 Do Proteins Have Chemical Groups
Other Than Amino Acids?
Proteins may be "conjugated" with other chemical
groups

• If the non-amino acid part of the protein is important to its

function, it is called a prosthetic group（輔基）.
• Be familiar with the terms: glycoprotein, lipoprotein,
nucleoprotein, phosphoprotein, metalloprotein,
hemoprotein, flavoprotein.
• Post-translational modifications are chemical changes
made to the protein after synthesis
5.7 Do Proteins Have Chemical Groups
Other Than Amino Acids?
5.7 Do Proteins Have Chemical Groups Other Than
Amino Acids?
Protein Structure and Function Are Tightly Linked

The three-dimensional structures of proteins and their

biological functions are linked by several overarching
principles（總體原則）:

• Function depends on structure

• Structure depends on sequence and on weak, noncovalent
forces（非共價力量）
• The number of protein folding patterns is large but finite
• Structures of globular proteins are marginally stable（臨界穩定
性）
• Marginal stability facilitates motion
• Motion enables function
What factors influence the folding process?

• Certain loci along the chain may act as

nucleation points（核心點，反應中樞）
• Protein chain must avoid local energy minima
• Chaperones may help
 6.3 What Are the Elements of Secondary Structure in
Proteins, and How Are They Formed?

• The atoms of the peptide bond ensemble lie in a plane

• Protein backbone structure is based on the amide plane
（醯胺平面）
• The resonance stabilization energy（共振穩定能量） of
the planar structure is 88 kJ/mol
• A twist about the C-N bond involves a twist energy of 88
kJ/mol times the square of the twist angle.
• Rotation can occur about either of the bonds linking the
alpha carbon to its two amide planes.
 6.3 What Are the Elements of Secondary Structure in Proteins,
and How Are They Formed?

Figure 6.2 The amide or peptide bond

planes are joined by the tetrahedral
bonds of the α-carbon.

The rotation parameters are φ and ψ.

The conformations shown corresponds
to φ= 180° and ψ= 180°.

Angle about the Cα-N bond is denoted φ (phi)

Angle about the Cα-C bond is denoted ψ (psai)
 Some Values of φ and ψ Are Not Allowed

Figure 6.3 Many of the possible conformations about an α-carbon

between two peptide planes are forbidden（禁止）because of steric
crowding（空間排擠）.
Steric Constraints on φ & ψ
• G. N. Ramachandran was the first to demonstrate
the convenience of plotting phi, psi combinations
from known protein structures
• The sterically favorable combinations are the basis
for preferred secondary structures

Figure 6.4 A Ramachandran diagram showing the

sterically reasonable values of the angles φ & ψ. The
shaded regions indicate favorable values of these
angles. Dots in purple indicate actual angles
measured for 1000 residues (excluding glycine, for
which a wider range of angles is permitted) in eight
proteins.
 Secondary Structure:
Regular Ways to Fold the Polypeptide Chain
(a)In the -helix the hydrogen bonds
are within a single polypeptide
chain and are almost parallel to the
helix axis.

(b) In the -sheet, the hydrogen bonds

are between adjacent chains and
are nearly perpendicular to the
chains.

(c) The 310 helix is found in proteins;

but, is less common than the -
-helix -sheet 310 helix helix.

Copyright © 2013 Pearson Canada Inc.

 The α-Helix
Numbers to Know
• Residues per turn: 3.6 amino acids

• Rise per residue: 1.5 Å (0.15 nm)

• Rise per turn (pitch): 3.6 x 1.5 Å = 5.4 Å

• The backbone loop that is closed by any H

bond in an alpha helix contains 13 atoms Linus Pauling with molecular model

• φ = −60 degrees, ψ = −45 degrees

 The α-Helix in Proteins

Figure 6.7 Two proteins that contain substantial amounts of α-helix.

 The β-Pleated Sheet
• The β-pleated sheet（摺板） is composed of β-strands（股）
• Also first proposed by Pauling and Corey, 1951
• Strands in a β-sheet may be parallel or antiparallel （對向平行）
• Rise per residue:
• 3.47 Å for antiparallel strands
• 3.25 Å for parallel strands
• Each strand of a β-sheet may be pictured as a helix with two
residues per turn
 The β-Pleated Sheet

Figure 6.10a. A “pleated sheet” of paper with an antiparallel β-sheet

drawn on it.
 The β-Turn

(aka β-bend, or tight turn)

• Allows the peptide chain to reverse direction
• Carbonyl C of one residue is H-bonded to the amide proton of a
residue three residues away
• Proline and glycine are prevalent in β-turns
• There are two principal forms of β-turns
 Helix-Sheet Composites in Spider Silk（蜘蛛絲）

Figure 6.11 Spider web silks are composites of α-helices and β-sheets.
The radial strands of webs must be strong and rigid: they have a higher
percentage of β-sheets. The circumferential strands (termed capture silk)
must be flexible; they contain a higher percentage of α-helices.
6.4 How Do Polypeptides Fold into Three-Dimensional Protein
Structures?

Several important principles:

• Secondary structures form wherever possible (due to formation of
large numbers of H bonds)
• Helices and sheets often pack close together
• Peptide segments between secondary structures tend to be short and
direct
• Proteins fold so as to form the most stable structures. Stability arises
from:
• Formation of large numbers of intramolecular hydrogen bonds
• Reduction in the surface area accessible to solvent that occurs upon folding
 6.4 How Do Polypeptides Fold into Three-Dimensional Protein
Structures?

• Two factors lie at the heart of these principles:

• Proteins are typically a mixture of hydrophilic and hydrophobic
amino acids
• The hydrophobic groups tend to cluster together in the
folded interior of the protein
 Fibrous Proteins（纖維蛋白）
• Much or most of the polypeptide chain is organized
approximately parallel to a single axis
• Fibrous proteins are often mechanically strong
• Fibrous proteins are usually insoluble
• Usually play a structural role in nature
• Three types of fibrous protein are discussed here:
• α-Keratin（角蛋白）
• β-Keratin
• Collagen（膠原蛋白）
 α-Keratin
• A fibrous protein found in hair, fingernails, claws, horns and
beaks
• Sequence consists of 311-314 residue-long alpha helical rod
segments capped with non-helical N- and C-termini
• Primary structure of helical rods consists of 7-residue repeats:
(a-b-c-d-e-f-g)n, where a and d are nonpolar （第一、四個氨基酸）.
• This structure promotes association of helices to form coiled
coils （盤繞線圈構形）
 α-Keratin

Figure 6.13 The structure of α-keratin.

 Fibroin and β-Keratin: β-Sheet Proteins

Proteins that form extensive beta sheets

• Found in and bird feathers

• Alternating sequence:
Gly-Ala/Ser-Gly-Ala/Ser....
• Since residues of a β-sheet extend alternately above and
below the plane of the sheet, this places all glycines on one
side and all alanines and serines on other side!
• This allows Glys on one sheet to mesh with Glys on an
adjacent sheet (same for Ala/Sers)
Fibroin and β-Keratin: β-Sheet Proteins

Figure 6.14 Silk fibroin consists of a stacked array of β-sheets.

 Collagen（膠原蛋白） – A Triple Helix

Principal component of connective tissue (tendons,

cartilage, bones, teeth)
• Basic unit is tropocollagen（原膠原）:
• Three intertwined polypeptide chains (1000 residues
each)
• MW = 285,000
• 300 nm long, 1.4 nm diameter
• Unique amino acid composition, including hydroxylysine
and hydroxyproline
• Hydroxyproline is formed by the vitamin C-dependent
prolyl hydroxylase（脯氨醯羥化酶） reaction.
 Collagen – A Triple Helix

Figure 6.15 Hydroxylation

of proline residues is
catalyzed by prolyl
hydroxylase.
 Collagen（膠原蛋白） – A Triple Helix
The features of its amino acid composition:
• Nearly one residue out of three is Gly
• Proline content is unusually high
• Collagen contains several unusual amino acids:
• 4-hydroxyproline
• 3-hydroxyproline
• 5-hydroxylysine
• Pro and HyPro together make 30% of residues
 The Collagen Triple Helix
A case of structure following composition

• The unusual amino acid composition of collagen is

unsuited for alpha helices or beta sheets
• It is ideally suited for the collagen triple helix: three
intertwined helical strands
• Much more extended than alpha helix, with a rise per
residue of 2.9 Å
• 3.3 residues per turn
• Long stretches of Gly-Pro-Pro/HyP
Collagen – A Triple Helix

Figure 6.16 Poly(Gly-Pro-Pro), a

collagen-like right-handed triple
helix composed of three left-handed
helical chains.
 Collagen Fibers

Staggered arrays of tropocollagens

• Banding pattern in electron micrographs with 68 nm repeat

• Since tropocollagens are 300 nm long, there must be 40-
nm gaps between adjacent tropocollagens (5 x 68 = 340
nm)
• 40-nm gaps are called "hole regions" - they contain
carbohydrate and are thought to be nucleation sites for
bone formation
 Collagen – A Triple Helix
Figure 6.17 In the electron microscope, collagen
fibers exhibit alternating light and dark bands.
The dark bands correspond to the 40-nm gaps
between pairs of aligned collagen triple helices.
 Globular Proteins Mediate Cellular Function

• Globular proteins are more numerous than fibrous proteins

• The diversity of protein structures in nature reflects the

remarkable variety of functions they perform

• Functional diversity derives in turn from:

• The large number of folded structures that polypeptides can adopt

• The varied chemistry of the side chains of the 20 common amino

acids
 Globular Proteins

Some design principles

• Helices and sheets make up the core of most globular proteins
• Most polar residues face the outside of the protein and interact
with solvent
• Most hydrophobic residues face the interior of the protein and
interact with each other
• Packing of residues is close
• However, ratio of vdW volume to total volume is only 0.72 to 0.77,
so empty space exists
• The empty space is in the form of small cavities（小腔室）
 Why does the protein core consist
primarily of α–helices and β–sheets?

• The protein core is predominantly hydrophobic

• The highly polar N-H and C=O moieties of the peptide

backbone must be neutralized in the hydrophobic core

• The extensively H-bonded nature of α-helices and β-

sheets is ideal for this purpose
 Waters on the Protein Surface Stabilize
the Structure
• The surface structure of a globular protein includes water
molecules
• The polar backbone and side chain groups on the protein
surface make H bonds with solvent water
• α-Helices on a protein surface are usually amphiphilic（兩性
特性）, with polar and charged residues facing the solvent
and nonpolar residues facing the interior
• A helical wheel （螺旋輪）presentation can reveal the
amphiphilic nature of an α-helix
• Some α-helices are hydrophobic and buried in the protein
interior
• Some helices are polar and entirely solvent-exposed
α-Helices May be Polar, Nonpolar
or Amphiphilic

Figure 6.22 The so-called helical wheel

（螺旋輪） presentation can reveal the
polar or nonpolar character of α-helices.
 Many proteins are composed of several
distinct domains（模組）

• Multidomain proteins typically are the sum of the functional

properties and behaviors of their constituent domains（模組）
• Proteins consisting of multiple domains probably evolved by the
fusion of genes（基因融合） that once coded for separate
proteins
• About 90% of domains in proteins have been duplicated（雙套化）
in other proteins
• Many proteins even contain multiple copies of the same domain
• Some of these often-duplicated domains
Many proteins are composed of several
distinct domains

Figure 6.25 Several protein modules used in the

construction of complex multimodule proteins.
Many proteins are composed
of several distinct domains

Figure 6.26 A sampling of proteins

that consist of mosaics of individual
protein modules（domains）.
 Denaturation Leads to
Loss of Protein
Structure and Function

• The cellular environment is suited to maintaining the weak forces that

preserve protein structure and function
• External stresses（外源壓力） – heat, chemical treatment, etc. – can
disrupt these forces in a process termed denaturation（變性） – the
loss of structure and function
• The cooking of an egg is an everyday example
• Ovalbumin（乳清蛋白）, the principal protein in egg white, remains in
its native structure up to a characteristic melting temperature, Tm
• Above this temperature, the structure unfolds and function is lost
 Figure 6.31 Proteins
can be denatured
(unfolded) by high
concentrations of
guanidine-HCl（鹽酸胍）
or urea（尿素）. The
denaturation of
chymotrypsin（糜蛋白酶）
is plotted here.

Protein 6.30 Proteins can be

denatured by heat, with
commensurate loss of function.
 The Protein Folding Energy Landscape
unfolded
Ken Dill has suggested that the folding
process can be pictured as a funnel（漏斗） Free energies high
of free energies（自由能）. The rim at the
top represents the many unfolded states.
Polypeptides ‘fall down the wall of the
funnel’ to ever fewer possibilities and lower
energies as they fold.

Folded Free energies

polypeptides low
Figure 6.34 A model for the
steps involved in the folding
of globular proteins.
 Marginal Stability of the Tertiary Structure
Makes Proteins Flexible

• A typical folded protein is only marginally stable（臨界穩定性）

• It is logical to think that stability is important to function, so why
are proteins often only marginally stable?
• The answer appears to lie in flexibility（靈活性） and motion
（運動性）
• It is becoming increasingly clear that flexibility and motion are
important to protein function
 Motion is Important for Globular Proteins

• Protein are dynamic structures – they oscillate（震盪） and

fluctuate（波動） continuously about their average or equilibrium
structures
• This flexibility is essential for protein functions, including:
• Ligand binding
• Enzyme catalysis
• Enzyme regulation

Figure 6.35 Proteins are dynamic

structures. The marginal stability of a
tertiary structure leads to flexibility and
motion in the protein.
 Molecular Chaperones Are Proteins That Help
Other Proteins to Fold
Why are chaperones（伴侶蛋白） needed if the information
for folding is inherent in the sequence?
• to protect nascent proteins（新生蛋白） during the
folding process and perhaps to accelerate slow steps
• Chaperone proteins were first identified as "heat-shock
proteins" (Hsp60 and Hsp70)
 Diseases of Protein Folding

• A number of human diseases are linked to abnormalities of protein

folding
• Protein misfolding（錯誤折疊） may cause disease by a variety of
mechanisms
• Misfolding may result is loss of function and the onset of disease
• The table on the next slide summarizes some known protein-folding
diseases
 Diseases of Protein Folding
澱粉樣蛋白

澱粉樣變性

普利昂蛋白

遺傳性肺氣腫

囊性纖維化
Thank You !