ANSWER KEY to Study Guide for Midterm 2
1. Self assembly systems are often pH sensitive. What do you think is the reason for this and which chemical groups are affected? What other factor(s) determine(s) the stability of protein complexes?
The pH of a solution affects the protonation state of acids and bases. In proteins it will affect lysine, arginine, glutamate, aspartate and specially histidine because His has a pKa value of 6 and is sensitive to changes close to physiological pH. Other factors are amphipathic structures, hydrogen bonds.
2. Explain how the specificity pocket of serine proteases affects the substrate-enzyme affinity, but not the kcat of the reaction. Begin your answer with the definition of the enzyme specificity and end with an argument in favor of renaming the specificity pocket the 'affinity' pocket.
The specificity pocket serves as a binding site for peptide substrates. This binding step precedes the formation of the transition state (tetrahedral intermediate) which is necessary for the catalytic process to occur. Therefore the specificity pocket affects the affinity of a substrate to the enzyme, but is not involved in the chemical reaction. The enzyme specificity is a term that is defined by the ratio of kcat/Km, while affinity only defines binding. The 'specificity' pocket therefore is better called affinity pocket.
3. Why are cystein residues in globular proteins advantageous for the study of protein folding pathways in vitro?
Because they allow the trapping of folding intermediates.
4. Which redox molecule in the photosynthetic reaction center couples electron and H+ transport?
Qb or ubiquinone. It is reduced twice before it transports protons across the cell membrane.
5. A Hill plot is used to determine if a protein shows cooperativity between subunits or not. How do you determine the Hill constant from the plot and how would the curve for a non-cooperative enzyme complex look like? Draw a Hill-plot for an enzyme with negative cooperativity.
The Hill coefficient is the slope of the binding curve in a double-logarithmic plot (the Hill plot) of oxygen binding at 50% binding (fractional saturation 0.5). A non-cooperative enzyme has a slope of 1, positive cooperativity a slope >1, and negative cooperativity a slope <1.
6. The melting curve of a protein shows a sigmoidal transition from the native fold to the unfolded conformation. What is the mechanism behind this type of curve? Give an other example of such a process for a macromolecule which is not a protein.
The sigmoidal shape of the melting curve indicates a cooperative process. The initial breaking of intramolecular interactions needs a high activation energy, but the subsequent unfolding is thermodynamically favored because of the rapid loss of hydrogen bonds and the stabilization of the denatured structure by hydrogen bonds with the solvent. An other example is DNA double strand denaturation.
7. Myoglobin and Hemoglobin belong to the same protein family. What level of structural information is usually used to define the evolutionary relationship between proteins? What other protein families do you know? Why can the tertiary structure for such analyses be more important than simple sequence?
Evolutionary relationship is measured by sequence comparison. Other protein families are nicotinic acetylcholine receptors, serine proteases, porins, reaction centers, carbonic anhydrase, K-channel, muscarinic acetylcholine receptor etc. . The structure of a protein is normally better conserved than the amino acid sequence because many similar residue can replace each other without distorting the tertiary fold. In addition, only the conformation of the active site needs to be conserved and often overall folds can vary substantially. If sequences are too dissimilar, it often helps to compare structures to establish evolutionary relationship.
8. How do the T and R conformation of the hemoglobin quaternary structure support a symmetric model of allostery?
T and R state are both stable structures as found by X-ray crystallography. The symmetric model assumes a coordinated switch (cooperativity) of all four subunits from a t to r states and back. This appears as a very good approximation to the 'real' situation.
9. What biological role(s) distinguishes Ca++ ions from other (divalent) metal ions?
It controls protein activity in dependence of its binding and unbinding equilibrium. The Ca free state is the inactive state, the Ca-bound state the active state. Many divalent cations are tightly bound and serve as active center in proteins (Zn, Fe, Mg).
10. Explain the name 'serine/aspartate/cystein protease' (consult the chapter on the HIV protease for aspartate proteases).
The names refer to the active amino acid residue of the proteolytic center.
11. You constructed a hydropathy plot of a not yet purified membrane protein (you know the sequence). You identified 6 hydrophobic stretches of at least 20 amino acids. What additional (structural) criterion do you need to know if those sequence fragments could be putative transmembrane spanning segments?
You need to predict the secondary structure and specifically if the segments are likely to adopt an alpha-helical conformation.
12. Explain the allosterism of the nicotinic acetylcholine receptor.
The ligand acetylcholine binds at the extracellular, N-terminal domain of two alpha subunits and this binding induces a conformational change within the membrane spanning part of the protein complex (the channel gate). Because ligand binding site and pore are located some 25angstrom apart, this is called an allosteric control mechanism.
13. Bacterial porin channels contain 16 anti-parallel beta strands that form a b barrel. What could you say about the distribution of amino acids along the sequence to make the channel stable in membranes an suitable for the flux of hydrophilic molecules?
For a beta barrel structure as an ion channel (porin subunits) the amino acids must be alternating polar and non-polar, thus creating an amphipathic beta sheet, with the barrel interior hydrophilic (ion pathway) and exterior hydrophobic (lipid anchor).
14. Why is the helix of gramicidin A called a beta helix? What is unusual about its amino acids?
It forms a wide helix with intramolecular hydrogen bonds similar to those found in beta sheets. The amino acids are alternating D- and L-amino acids.
15. What is the driving force of self-assembly systems / protein-folding in water?
The hydrophobic effect.
16. What are the basic concentration conditions of enzyme and substrate to measure Vmax of enzymatic catalysis?
Vmax can only be established if the product rate formation is measured at substrate concentrations exceeding the enzyme (active site) concentration. This is called saturation.
17. Explain one possible way of lowering the activation energy for an enzymatic reaction.
Apposition of the reactant substrates in a conformationally optimal way.
18. How does the tertiary structure of the chymotrypsin family of serine proteases affect the kcat of the proteolytic activity?
It does not affect the kcat, nor Km. This is only determined by the active site conformation.
19. You analyzed the specificity pocket of a new type of protease and found Lysine as part of it. What could you say about the sequence specificity of the protease?
Residue Rn-1 on the substrate is likely to be glutamate or aspartate.
20. What is the reason to find amphipathic helices on the surface of proteins/at the center of ion channel complexes like the acetyl cholin receptor?
The hydrophilic side of the helix is oriented toward the water exposed surface/channel and the hydrophobic side towards the interior which is often a hydrophobic core to stabilize the overall structure of a protein in aqueous solution.
21. Ion channels can be compared to enzymes, even though they do not catalyze a chemical reaction? Describe the enzymatic mechanism of a ligand gated ion channel. What is the role of allostery in ligand gated channels? (see #12)
Ion channel provide binding sites for ions and often provide ion selectivity besides the obvious hydrophilic pathway for charged molecules across the hydrophobic membrane. They serve as conductors in an electrically insulating material.
22. Thermal motion is important in enzymatic activity? Explain.
Substrate and product need to diffuse into and out of active sites. Residues on enzymes often are flexible and thus provide transition state stabilization (induced fit binding). Every system has different 'needs' for thermal motion. The catalytic triade of serine proteases depends on a reduced flexibility of its serine and histidine residues for optimal interaction with the scissile bond (reducing the activation energy for the nucleophilic attack).