Chapter Protein Structure

e14
and Function

Levels of Protein Structure......e280

Some Proteins Are

Nanomachines...........................e282 Levels of Protein Structure
DNA-Binding Proteins...............e283 Proteins constitute a majority of the dry weight for cells. They
are extremely versatile, having various roles in the cells, including
enzymes, structural, communication, transport, etc. The function of the
protein is directly related to its structure. This chapter focuses on the
relationship between structure and function. The Key Concepts are
described below.

l The primary structure of proteins is the amino acid sequence of

the polypeptide chains.
l The secondary structure of proteins results from hydrogen

bonds.
l The tertiary structure of proteins is due to interactions between

amino acid side chains. Larger proteins may have multiple fold-
ing domains.
l The quaternary structure of proteins refers to the assembly of

multiple polypeptide chains to form proteins with subunits.

The structure of a protein is often the result of multiple inter-

actions at four different levels: primary, secondary, tertiary, and
quaternary.
The primary structure of a protein is simply the sequence of
amino acids. This structure is dictated by the information encoded with
DNA and the mRNA copy.
The secondary structure of proteins results from hydrogen bond-
ing occurring between the various functional groups present in the
backbone of the polypeptide. Several secondary structures may occur
but the two most common are alpha-helices and beta-sheets. The
α-helices are coiled structures with hydrogen bonds between adja-
cent peptide backbones and running in parallel to the helix core.
The β-sheets form from the folding of the polypeptide chain back
upon itself to yield a zigzag shape. Hydrogen bonds once again form
between functional groups present in the backbone.

Molecular Biology, Second Edition Study Guide.

© 2013 Elsevier Inc.
e280 Academic Cell is an imprint of Elsevier Inc.
 Levels of Protein Structure e281

The tertiary structure is due to interactions among the variable groups of each
amino acid. Hydrophobic interactions play a role at this structural level. Amino acid
residues that are hydrophilic (water-loving) prefer to be on the outside of the protein
and in contact with water. In contrast, hydrophobic (water-fearing) residues prefer
the inside of the protein away from water. In addition to hydrophobic interactions,
other forces are also at work to maintain the 3-D structure of proteins, which include
hydrogen bonds, van der Waals forces, disulfide bonds, and ionic bonds. Hydrogen
bonds may form between adjacent amino acid residues. Van der Waals forces
are weak interactions among complementary-shaped residues. That is, the shapes
must fit together for van der Waals to affect them. Covalent disulfide bridges can
link adjacent cysteine residues together, which increases the stability of the protein.
Finally, ionic interactions occur when opposite charges on amino acids are near to
each other.
Quaternary structure forms from the assembly of multiple, independent polypep-
tide chains. These associations are mostly due to hydrophobic interactions. The subu-
nits in the quaternary structure may be multiple copies of the same polypeptide or
may constitute several different polypeptide sequences.

l Protein structures are investigated using X-ray crystallography, NMR spectro­

scopy, and the electron microscope.

The structure of proteins can be elucidated using several advanced techniques.

X-ray crystallography relies on the acquisition of purified protein that is crystalized.
Any impurities will likely interfere with crystallization. An X-ray is passed through
the crystal of purified protein and the diffraction pattern is examined. From the pat-
tern, the 3-D shape can be determined. X-ray crystallography was also used to deter-
mine the conformation of DNA, a nucleic acid.
Nuclear magnetic resonance (NMR) spectroscopy bombards proteins with
radio waves, which cause the nuclei of the atoms to align and absorb energy. The
energy is then analyzed by a computer to determine the composition and location of
the atoms.
The electron microscope is a high powered microscope that uses electrons as the
illuminating agent. The electron microscope is limited in its ability to visualize the
interior of a protein but is very powerful in its assessment of the protein’s surface.

l Proteins often contain other chemical components in addition to the polypep-

tide chains. These may include metal ions, organic co-factors, and attached
sugar and lipid residues.

For functionality, other chemical components associate with the polypeptide

chains. These components include cofactors and coenzymes, and modifications by
addition of carbohydrates and lipids.
Cofactors include metal ions that are coordinated by specific amino acid residues
in the polypeptide chain. Common metal ions include magnesium, zinc, copper, man-
ganese, and iron, among many others.
Coenzymes are not enzymes, but rather organic cofactors. These include many
vitamin derivatives, such as vitamins A, C, and D. Additionally, they may include
nucleotide derivatives, such as nicotinamide adenine dinucleotide (NAD) and flavin
adenine dinucleotide (FAD). These coenzymes function in redox reactions by carry-
ing high energy electrons.
Proteins may also have other groups attached to them. Glycoproteins are
polypeptides containing post-translationally attached carbohydrates. Lipoproteins
are polypeptides with attached lipids. The addition of these attached substances aids
in the function of the protein. For example, glycoproteins are usually localized to the
cell surface and aid in cell-to-cell adhesion and recognition. Lipoproteins might be
attached to the cell surface for an external function.
e282 CHAPTER FOURTEEN • Protein Structure and Function

Some Proteins Are Nanomachines

l Proteins comprise about 60% of the organic matter of a typical cell and
carry out many functions. Complex protein assemblies may be regarded as
nanomachines.

Proteins serve a variety of roles inside the cell. They may carrying out biochemi-
cal reactions or may serve a structural or transport role. Assemblies of many proteins
are regarded as nanomachines because of their complex roles in the cell. Examples of
nanomachines include the DNA replication complex, RNA spliceosome, and protein
degradation complex.

Maillard RA, et al. (2011) ClpX(P) Generates Mechanical Force to Unfold of target proteins. ClpX then unfolds and moves the protein towards ClpP, the pepti-
and Translocate Its Protein Substrates. Cell 145:459-469. dase. In this paper, the authors demonstrate that ClpX exerts a mechanical force
upon the target protein to unfold it.
Proteases are often sequestered in large complexes away from Using optical tweezers in conjunction with a single molecular assay
other cellular proteins to prevent their uncontrolled degradation of proteins. The system, the authors were able to determine that protein denaturation was induced
peptidase complex for ATP-dependent proteases consists of several different pro- by ClpX through a power-stroke mechanism to mechanically force the unfolding of the
polypeptide. The target polypeptide used in this experiment was green fluorescent pro-
tein (GFP). Furthermore, ClpP aided in unfolding by reducing slippage by ClpX. After
several attempts, ClpX was finally able to unravel the β-sheets of GFP, after which the
protein spontaneously unraveled, and was translocated to ClpP for proteolysis.
FOCUS ON
RELEVANT RESEARCH Conceptual questions
1. What are nanomachines?
2. Why does this associated paper fall under the Key Concept about nanomachines?
3. Besides protein degradation, what other functions do nanomachines serve?
teins and enzymes that are responsible for protein degradation arranged in a barrel
conformation. This complex is also an example of a cellular nanomachine. During
Discussion points
protein degradation, peptidases associate with ATP-dependent unfoldases to unfold
and translocate the protein into the chamber for degradation. The higher level protein Proteins represent a majority of cellular organic mass and consume
structures are removed so that only the primary structure remains and is targeted by large amounts of resources and energy during their synthesis. Why is it important
the proteolytic enzymes. to have degradation nanomachines available for proteins after the expense of
The authors of this associated paper characterize the role of ClpX making them? In what situations might the cell require less proteins, particularly
from Escherichia coli. ClpX is an ATPase that recognizes an ssrA tag on the C-terminus specific ones?

l Enzymes are proteins that catalyze chemical reactions. They act by lowering
the energy of activation.

Enzymes are a specific type of protein within the cell that can catalyze chemical
reactions. This catalysis is due in part to an enzyme’s ability to lower the energy of
activation for a chemical reaction. This means that less energy is required to start the
reaction.
Examples of enzymes include the light-emitting luciferase, the ATP synthesizing
ATP synthase, protein degradation enzymes called proteases, and enzymes involved
in large biochemical pathways such as respiration, fermentation, or photosynthesis.

l Different enzymes bind their substrates with different specificities.

The chemical substance being acted upon by an enzyme is the substrate. The
shape and conformation of the enzyme’s active site (the location of the chemical
 DNA-Binding Proteins e283

reaction on the enzyme) ultimately determines the binding specificity of the enzyme.
For instance, the enzyme β-galactosidase has an affinity for its substrate lactose. This is
a very specific interaction as the enzyme will not bind other disaccharides.

l The rate of enzyme reactions depends on the substrate concentration, the

affinity of the enzyme for the substrate, and the inherent properties of the
active site (reflected by the Vmax).

Reaction rates are proportional to the amount of substrate present, as long as the
concentration is low, and available to the enzyme. Higher amounts of substrate satu-
rate the enzyme, which works no faster at even higher substrate concentrations. This
is the maximum velocity, or Vmax (Vm) of the enzyme. The Vm is also dependent upon
temperature, pH, and enzyme concentration.

l Some enzymes are directly regulated by binding small signal molecules, others
are controlled by chemical modification.

Even though enzymes are usually quite specific for their substrates, other chemi-
cals that resemble the natural substrates may also bind the enzyme’s active site. These
analogs often regulate the enzymatic activity by competitively inhibiting the enzyme,
since they compete with the natural substrate for the active site. The binding of the
inhibitor may be either temporary or permanent, resulting in the shutdown of the
enzyme reaction.
Biochemical pathways are regulated by a process called negative feedback. When
the end product of the pathways accumulates to sufficient concentrations, the product
binds to and inactivates the first enzyme in the pathway, essentially shutting down the
pathway to prevent a waste of resources.
Allosteric sites are second binding sites on enzymes where chemical reactions do
not occur. These sites play a role in protein regulation by altering the conformation of
the protein to a form that can readily accept a substrate or reject a substrate.
Chemically modifying enzymes is also a method the cell uses to regulate protein
function. Additions of phosphate groups are common modifications, but other modi-
fications may occur. These include addition of methyl, acetyl, and adenyl groups. The
addition or subtraction of these groups modulates enzyme action by either inhibition
or activation, depending upon the specific enzyme.

DNA-Binding Proteins
l Some proteins bind to DNA at specific sequences. Several different structural
motifs are common in DNA-binding proteins.

Various proteins bind in the major groove of DNA molecules and perform a spe-
cific function. Examples of DNA-binding proteins include those involved in replica-
tion, transcription, gene expression regulation, and DNA repair, among many others.
Although there is great diversity in the structure and function of DNA-binding proteins,
some polypeptide motifs enable the binding to the major groove of DNA. These motifs
include helix-turn-helix (HTH), helix-loop-helix (HLH), zinc fingers, and leucine zippers.
HTH and HLH are similar in that they both contain two α-helices separated by a
turn (short) and a loop (long) region. Both of these usually bind as dimers to inverted
repeats in the DNA sequence.
Leucine zippers are α-helices that contain a leucine residue every seventh amino
acid. This motif is found in many eukaryotic transcription factors. Zinc fingers con-
sist of 25-30 amino acids surrounding a single zinc atom, which is coordinated by two
cysteines, which are very close to short α-helices. It is the helices that make contact
with the DNA.
l Proteins may be denatured by heat, detergents, and assorted chemicals.
e284 CHAPTER FOURTEEN • Protein Structure and Function

Lammens K, et al. (2011) The Mre11:Rad50 Structure Shows an The results of their experiment indicate that Mre11 and Rad50 form
ATP-Dependent Molecular Clamp in DNA Double-Strand Break Repair. Cell a large clamp that recognizes double-stranded DNA breaks. Furthermore, they were
145:54-66. able to show that ATP binding to Rad50 induced a change in the conformation of
the Mre11 dimer. This suggests a role for the ATP-binding by Rad50 in controlling
Many motifs are involved in the binding of DNA. These motifs Mre11’s processing of DNA. The authors discovered that when Rad50 is bound to
include the helix-turn-helix (HTH), helix-loop-helix (HLH), zinc fingers, and leucine ATP, the crosslink at Mre11’s dimer interface becomes altered and DNA-binding occurs
more tightly.

Conceptual questions
FOCUS ON
1. Besides histones, what types of proteins bind to DNA?
RELEVANT RESEARCH 2. What are some examples of DNA-binding motifs?
3. Where do most DNA-binding motifs bind the DNA?

zippers. The associated paper focuses on the roles of the HLH motif of Mre11. Mre11 Discussion points
is involved in the response of Saccharomyces cerevisiae, a yeast, to double-stranded
When investigating a new protein, often researchers use computer
DNA breaks. Two Mre11 associate with two Rad50 to form a large MR complex
programs to propose a tertiary structure based upon the known amino acid sequence.
that contains both ATP-stimulated nuclease activity and DNA-binding capabilities. The
If the protein of interest is suspected of binding to DNA, what types of features would
authors investigated the structural framework for Mre11 and Rad50 interaction using
you look for in the tertiary structure? Do you think that it is difficult to predict three-
X-ray crystallography of the catalytic portion of the MR complex from Thermotoga
dimensional structures of proteins? Why?
maritima. Additionally, the interactions of the HLH motif from Mre11 with Rad50 was
also investigated by X-ray crystallography.

Denaturation is the loss of a protein’s 3-dimensional shape due to changes in the

noncovalent interactions. Once the 3D shape is lost, function usually follows. Proteins
can be denatured by addition of detergents and chemicals, along with changes in tem-
perature. Detergents disrupt the hydrophobic interactions necessary to maintain the
tertiary structure. Chemicals such as β-mercaptoethanol disrupt disulfide bonds, thus
destabilizing the protein. Heat denatures the hydrogen bonds that hold the secondary
structures together. Although denaturation means a loss in 3D shape for the protein,
it does not mean that the primary structure is also lost. The sequence of amino acids
remains in a denatured protein.