Bke2 Biochemistry Exercises

Suggested answers to Group Excercise: Protein structure and function

Name four types of functions proteins can have in cells, and outside them. Think of at least one example for each case.

Answer:
Proteins have many functions. Some examples are: (Horton Ch 3 p 51-52)

Enzyme catalysis: speed up chemical reactions. e.g. proteolytic enzymes
Transport. e.g. in blood, hemoglobin carries oxygen
Storage. e.g. seeds, egg white
Motion. e.g. myosin in muscles, flagella
Mechanical support. e.g. collagen in skin and bone
Immune defence e.g. antibodies
Regulation of various functions e.g. nerve impulses, receptors, hormones & transcription factors

On the ribosome, amino acids are covalently linked together to form a polypeptide chain. Draw a tripeptide, and label the important parts (C-alpha, main chain, side chain, peptide bond, termini). Indicate which bonds are free to rotate. What determines the sequence of amino acid residues found? Why is this sequence important?

Answer:
A polypeptide chain starts with an amino terminus (N-terminus) and ends with a carboxyl-terminus (C-terminus). In between there are a number of amino acid residues linked together covalently, the junctions forming rigid and planar peptide units. One main chain carbon atom (named Ca) is attached to the variable side chain R. Rotation of the main-chain bonds next to the Ca atom is possible (angles named y and f). There are ~20 different commonly used side chains which vary in size, charge, hydrogen bonding capacity and chemical reactivity. Many of their bonds are free to rotate, as well. The sequence of the amino acid residues in a protein is determined by the sequence of the bases in the corresponding gene (DNA). The sequence of the amino acid residues determine the three dimensional structure, and the three dimensional structure determines the function. (Horton 3.5 p64-66, 4.3 p86-88)

During or after synthesis, a polypeptide folds to form a functional protein. What do we mean by the term "folding"? Give a short description of the process, and the forces that make it happen.

Answer:
Protein folding is the process of making a compact three dimensional structure from the linear polypeptide chain. Folding may start even before the synthesis of the polypeptide chain has been fully completed on the ribosome. Secondary structural elements form early (mostly based on hydrogen bonding). These then pack against each other in ways that bury hydrophobic side chains in the interior of the protein (i.e. form the hydrophobic core), and let hydrophilic residues either interact with each other, or lie on the surface where they can interact with water. (In fact, burying hydrophobic side chains provides the main driving force of folding.) During the process, various intermediates are formed, for which the structures become more and more stable. In many proteins the folded structure is further stabilised by disulfide bridges. The final structure, which we consider the native form, is usually the most stable and thermodynamically favorable structure, and is most often that associated with function. (Horton 4 p81-83, 4.10 p107-112)

List the amino acid side chains most frequently found in the cores of folded protein. What do they have in common? What types of amino acids are most often found on the surface? What features do they have in common?

Answer:
Ala, Val, Leu, Ile, Phe, Met, Cys, Gly are most common in the cores of proteins. These residues are aliphatic or aromatic, and thus non-polar (hydrophobic). Disulphide bonds (involving two Cys residues) also cause these residues to be buried.
Residues that are polar or charged (e.g. Asp, Glu, Arg, Ser and others) are most common on the surface of the protein. (Horton 3.2 p52-59, 4.10 p107-111)

Compare the interactions (hydrogen bonding, van der Waals, etc.) that are found in an unfolded protein to those of a correctly folded protein. What does this suggest about the basis of the stability of folded proteins?

Answer:
In a fully unfolded protein the interactions between side chains far apart in the sequence are very few, while in folded proteins, many result from the more compact arrangement.
Hydrogen bonds. In an unfolded protein, hydrogen bonds are formed between polar groups and water molecules. In a folded molecule the hydrogen bonds instead are formed between polar groups within the protein (main chain as well as side chains). Note that essentially all atoms that have a hydrogen bonding capacity do form hydrogen bonds - with whatever is available (protein or H₂O in the solution).
Interactions between hydrophobic residues. Hydrophobic residues in the folded protein pack in the interior of the molecule, away from the polar environment of the surrounding solution. These residues, as well as any others that are in close contact with each other (within 4 Å or so) interact through van der Waals interactions.
van der Waals interactions. Side chains from different regions of the folded polypeptide chain come close and are held together by van der Waals forces. Close contacts is needed for van der Waals forces.
Electrostatic interactions are also formed when charged groups from different regions come together upon folding.

A folded protein is stabilized by a collection of many weak forces that together are strong. Placing hydrophobic side chains together in a non-polar environment is the major energy advantage of the folded state.
(Horton 4.10 p107-110)

Name the most common kinds of secondary structural elements, and describe how they are held together. Define the terms tertiary structure and quaternary structure. How are these held together? Why are these things important?

Answer:
Secondary structural elements are repeated arrays held together by hydrogen bonds. The most common types are:

a-helix: right-handed helix. There is a hydrogen bond between the carbonyl oxygen of an amino acid and the amide nitrogen four residues further along the sequence. A helix therefore contains a series of relatively local arrangements that overlap.
b-strands/sheets: extended chains running parallel or anti parallel, with main chain hydrogen bonds between the strands. The resulting sheet is often more or less twisted, and can involve strands found quite distant in the sequence.

Tertiary structure is the arrangement of secondary structural elements in three-dimensional space; residues far apart in sequence are brought together; some cysteines are bound to form disulphides.
Quaternary structure is the arrangement of multiple subunits (e.g. the four subunits of hemoglobin), i.e. an interaction involving more than one polypeptide chain. Most proteins are made up of only one chain, so have no quaternary structure.
Tertiary and quatarnary structure are both held together by hydrophobic interactions as well as hydrogen bonds. These things are important, because they help use describe what proteins look like, and that is very much a part of how they do their job.
(Horton 4.4-4.8 p89-104)

Many cosmetic creams claim to contain collagen as an active ingredient. What does collagen look like? What would be the technical problems of actually including collagen in such a cream? Would it be likely to help your skin?

Answer:
As a result of its high content of proline and hydroxyproline, collagen makes a special kind of left-handed helix. Three of these helices wrap around each other to make a supercoil that is quite stable and insoluble. Collagen is very difficult to hydrolyze (and so solubilize) to make it available in a cream. Even if you can get it soluble, there is no real way of getting it back into the collagen structure that is also found in your skin. So, collagen is unlikely to help your skin, and may even trigger an allergic reaction. (Horton 4.11 p113-114)

Which of the following would be expected to stabilize the structure of a protein? Why?
a) introduction of disulfide bonds
b) substitution of a pair of amino acid residues that are hydrogen bonded to each other with a pair of glycine residues
c) replacement of a small amino acid in the crowded hydrophobic core of a protein with Trp)
d) decrease in the number of Pro and hydroxy-Pro residues in a collagen molecule
Think of a way you might measure the extent of the change in stability.

Answer:
Introduction of properly placed disulfide bonds could stabilize a protein (Horton 3.2C p57). Removal of hydrogen bonds (Horton 4.10B p109), or introduction of steric clashes (Horton 4.10A p108-109, will de-stabilize them. Collagen structure depends on having Pro and hydroxy-Pro residues in the right places, so their loss would be destabilizing (Horton 4.11 p113-114).
You might measure differences, for example, by comparing the different proteins' resistance to thermal or chemical denaturation. Spectroscopic methods often can provide a sensitive measure of proper folding. (Horton 4.9 p105-106)

You have isolated a protein that binds and acts on a negatively-charged substrate, and determined by peptide mapping that the key part falls somewhere in the following portion of the sequence:
ALMSQRTWNGKCP
Which residue or residues would you test first, if you were going to make mutants of the protein?

Answer:
You should mutate the R (arginine) and K (lysine) residues first, and in separate experiments. Each has a positive charge, and therefore either (or both) might be involved in binding a negatively-charged substrate.

Why do our cells use proteins that bind heme for oxygen transport, rather than just using heme directly? What aspects of the proteins are most important in making the protein-heme partnership work?

Answer:
Heme by itself in solution will bind oxygen, but it is too prone to oxidation (and inactivation), and also will not effectively prefer oxygen to other ligands. In the protein, the proper hydrophobic environment provides a binding site for heme. A pocket is found in the protein immediately next to the important iron of the heme. This pocket is designed to promote the binding of oxygen, rather than carbon monoxide, for example. Thus heme bound to protein will have special characteristics resulting from the environment the protein has designed for it. (Horton 4.13A p116)

What does the term "allostery" mean? What are the general roles of allostery in the function of hemoglobin and myoglobin? What general features of a protein are needed for allostery to work?

Answer:
The word allosteric comes from the Greek words meaning "other shape". It means that the protein has (at least) two different shapes (conformations) that have different functional properties. The relative amounts of the two states can be changed by binding things at the active, and other, sites. So, binding at one site can change the properties of another site on the protein. Allosteric interactions which involve two different kinds of sites are termed heterotropic allosteric interactions; they may occur within the same molecule. Allosteric proteins often have more than one subunit, and so they also can generally communicate this kind of information from one active site to that on another subunit (also through conformational changes). This is called cooperativity, or homotropic allosteric interactions. Allosteric effects on activity may be positive or negative. The allosteric properties of hemoglobin allow its activity to be controlled, unlike the simpler case of myoglobin. This allows the arrangement of oxygen transport so that oxygen is efficiently picked up at the lungs, and delivered to the tissues as needed. For allostery to work, there must be multiple binding/active sites, and a way to change the proteins shape so that these sites can communicate with each other. (Horton 4.13 p116-121)

A fetus is absolutely dependent on its mother for oxygen. How do you think a fetus' hemoglobin will differ from that of its mother?

Answer:
Fetal hemoglobin is able to bind oxygen more tightly than maternal hemoglobin can under physiological conditions. The substitution of a gamma chain for a beta chain gives the fetal hemoglobin a lower affinity for BPG, and thus a higher affinity for oxygen. Where the two hemoglobins are competing, like in the placenta, fetal hemoglobin will win the race, and become oxygenated.

Why is the pH relatively low in the capillaries? What consequences does that have for oxygen transport? When you get out of breath during strenuous excercise, lactic acid builds up in the muscles. What effect would that have on your oxygen supply?

Answer:
In the tissues, CO₂ is produced during metabolism, and the pH is lowered; this is easily carried over to the nearby bloodstream. The Bohr effect describes how low pHs and high [CO₂] change hemoglobin to lower its oxygen affinity. Myoglobin is not so affected, so it will "profit" when hemoglobin sets the oxygen free. If the pH is lowered further, as when lactic acid builds up, even more oxygen will become available to myoglobin. (Horton 4.14C p121)