Protein Three-Dimensional Structure: Problems

The energy barrier that must be crossed to go from the polymerized state to the hydrolyzed state is large even though the reaction is thermodynamically favorable.

Matters of stability. Proteins are quite stable. The lifetime of a peptide bond in aqueous solution is nearly 1000 years. However, the free energy of hydrolysis of proteins is negative and quite large. How can you account for the stability of the peptide bond in light of the fact that hydrolysis releases considerable energy?

(A) TEPIVAPMEYGK
(B) -1 at pH 7
(C) -4 at pH 12

First abbreviate, then charge. Examine the following peptide and answer.
Thr-Glu-Pro-Ile-Val-Ala-Pro-Met-Glu-Tyr-Gly-Lys
(A) Write the sequence using one-letter abbreviations.
(B) Estimate the net charge at pH 7.
(C) Estimate the net charge at pH 12.

This observation demonstrates that pKa values are affected by the environment. A given amino acid can have a variety of pKa values, depending on the chemical environment inside the protein.

Neighborhood peer pressure? More than 500 pKa values have been determined for individual groups of proteins. Account for this discrepency.

The peptide bond has partial double-bond character, which prevents rotation. This lack of rotation constrains the conformation of the peptide backbone and limits possible structures.

Why is rotation about the peptide bond prohibited, and what are the consequences of the lack of rotation?

Sequence of amino acids in a protein

Primary structure

The bond responsible for primary structure

Peptide (amide) bond

Forms between two cysteine atoms

Disulfide bond

Angle of rotation about the bond between the N atom and the α-carbon atom

phi angle

Angle of rotation between the α-carbon atom and the carbonyl carbon atom.

psi angle

A plot of phi and psi angles

Ramachandran diagram

A rodlike structure with a tightly coiled backbone

α helix

Formed by hydrogen bonds between parallel or antiparallel chains

ß pleated sheet

Fully extended polypeptide chain

ß strand

Regular repeating 3-D structures

Secondary structure

20^50, or 1.13x10^65, a very large number

Alphabet soup. How many different polypeptides of 50 amino acids in length can be made from the 20 common amino acids?

The (nitrogen-α carbon-carbonyl carbon) repeating unit.

Vertebrate proteins? What is meant by the term polypeptide backbone?

Side chain is the functional group attached to the α-carbon atom of an amino acid.

Not a sidecar. Define the term side chain in the context of amino acid or protein structure.

Amino acid composition refers simply to the amino acids that make up the protein. The order is not specified. Amino acid sequence is the same as the primary structure- the sequence of amino acids from the amino terminal to the carbonyl terminal of the protein. Different proteins may have the same amino acid composition, but sequence identifies a unique protein.

One from many. Differentiate between amino acid composition and amino acid sequence.

The primary structure determines the tertiary structure. Knowing it helps to elucidate the function of the protein.

List some of the benefits of knowing the primary structure of a protein.

The helix is a condensed, coiled structure, with the R groups bristling outward from the axis of the helix. The distance between two adjacent amino acids is 1.5 Å. The strand is a fully extended polypeptide chain and the side chains of adjacent amino acids point in opposite directions. The distance between adjacent amino acids is 4.5 Å. Both structures are stabilized by hydrogen bonding between components of the polypeptide backbone.

Compare and contrast. List some of the differences between an α helix and a ß strand.

Primary structure - peptide bond; secondary structure - local hydrogen bonds between components of the polypeptide backbone; tertiary structure - various types of noncovalent bonds between R groups that are far apart in the primary structure; quaternary structure - various noncovalent bonds between R groups on the surface of subunits.

Degrees of complication. What are the levels of protein structure? Describe the type of bonds characteristic of each level.

Refers to the spatial arrangement of amino acid residues that are far apart in the sequence

Tertiary structure

Combinations of secondary structure that are present in many proteins

Supersecondary structure

Compact regions that may be connected by a flexible segment of polypeptide chain

Domain

Basic component of quaternary structure

Subunit

Refers to the arrangement of subunits and the nature of their interactions

Quaternary structure

An energy landscape

Folding funnel

Structure characterized by dynamic hydrophobic interactions

Molten globule

Proteins that exist in an ensemble of structures of approximately equal energy that are in equilibrium

Metamorphic protein

Proteins that in whole or in part lack discrete 3-D structure under physiological conditions

Intrinsically unstructured protein

Cause of spongiform encephalopathies

Prion

No, the Pro-X bond would have the characteristics of any other peptide bond. The steric hindrance in X-Pro arises because the R group of Pro is bonded to the amino group. Hence, in X-Pro, the proline R group is near the R group of X, which would not be the case in Pro-X.

Who goes first? Would you expect Pro-X peptide bonds to tend to have cis conformations like those of X-pro bonds? Why or why not?

The methyl group attached to the ß-carbon atom of isoleucine sterically interferes with α-helix formation. In leucine, this methyl group is attached to the y-carbon atom, which is farther from the main chain and hence does not interfere.

Contrasting isomers. Poly-L-leucine in an organic solvent such as dioxane is α helical, whereas poly-L-isoleucine is not. Why do these amino acids with the same number and kinds of atoms have different helix-forming tendencies?

The first mutation destroys activity because valine occupies more space than alanine does, and so the protein must take a different shape, assuming that this residue lies in the closely packed interior. The second mutation restores activity because of a compensatory reduction of volume; glycine is smaller than isoleucine.

Active again. A mutation that changes an alanine residue in the interior of a protein into valine is found to lead to a loss of activity. However, activity is regained when a second mutation at a different position changes an isoleucine residue into a glycine. How might this second mutation lead to a restoration of activity?

Loops invariably are on the surface of proteins, exposed o the environment. Because many proteins exist in aqueous environments, the exposed loops are hydrophilic so as to interact with water.

Exposure matters. Many of the loops on the proteins that have been described are composed of hydrophilic amino acids. Why?

(A) Heat would increase the thermal energy of the chain. The weak bonds holding the chain in its correct 3-D structure would not be able to withstand the wiggling of the backbone and the tertiary structure would be lost. Often, the denatured chains would interact with each other, forming large complexes that precipitate out of solution.
(B) Detergents would denature the protein by essentially turning it inside out. The hydrophobic residues in the interior of thee protein would interact with the detergent, whereas the hydrophilic residues would interact with one another and not with the environment.
(C) All ionic interactions, including hydrogen bonds, would be disrupted, resulting in protein denaturation.

Goodbye native state. Hello chaos. How would each of the following treatments contribute to protein denaturation?
(A) Heat
(B) Addition of the hydrophobic detergents
(C) Large changes in pH

Glycine has the smallest side chain of any amino acid. Its size is often critical in allowing polypeptide chains to make tight turns or to approach one another closely.

Often irreplaceable. Glycine is a highly conserved amino acid residue in the evolution of proteins. Why?

Glutamate, aspartate, and the terminal carboxylate can form salt bridges with the guanidinium group of arginine. In addition, this group can be a hydrogen-bond donor to the side chains of glutamine, asparagine, serine, threonine, aspartate, glutamate, and tyrosine and to the main-chain carbonyl group. At pH 7, histidine also can hydrogen bond with arginine.

Potential partners. Identify the groups in a protein that can form hydrogen bonds or electrostatic bonds with an arginine side chain at pH 7.

Disulfide bonds in hair are broken by adding a thiol-containing reagent and applying gentle heat. The hair is curled, and an oxidizing agent is added to re-form disulfide bonds to stabilize the desired shape.

Permanent waves. The shape of hair is determined in part by the pattern of disulfide bonds in keratin, its major protein. How can curls be induced?

Some proteins that span biological membranes are "the exception that prove the rule" because they have the reverse distribution of hydrophobic and hydrophilic amino acids.

Location is everything 1. Most proteins have hydrophilic exteriors and hydrophobic interiors. Would you expect this structure to apply to proteins embedded in the hydrophobic interior of a membrane? Explain.

The amino acids will be hydrophobic in nature. An α helix is especially suitable for crossing a membrane because all of the amide hydrogen atoms and carbonyl oxygen atoms of the peptide backbone take part in intrachain hydrogen bonds, thus stabilizing these polar atoms in a hydrophobic environment.

Location is everything 2. Proteins that span biological membranes often contain α helices. Given that the insides of membranes are highly hydrophobic, predict what types of amino acids will be in such a helix. Why is an α helix particularly suitable for existence in the hydrophobic environment of the interior of a membrane?

Recall that hemoglobin exists as a tetramer, whereas myoglobin is a monomer. Consequently, the hydrophobic residues on the surface of hemoglobin subunits probably take part in van der Waals interactions with similar regions of the other subunits and are shielded from the aqueous environment by these interactions.

Greasy patches. The α and ß subunits of hemoglobin bear a remarkable structural similarity to myoglobin. However, in the subunits of hemoglobin, residues that are hydrophilic in myoglobin are hydrophobic. Why?