(15) 1a. Draw the structure of thefollowing peptide at pH 7 (structures of aa side- chains are on sale for 1 pt each!):
(10) b. Draw a titration curve for a peptide having the same amino acid residues as (a) above from pH 2-12 (assume that it is soluble) indicating approximate pKavalues. Label axis, buffer regions, and approximate pKa's!
(10) 2. Discuss the concept of domains in protein structure. Include in your discussion the relation between genes and domains, the potential consequences for adaptive evolution, and the effects/consequences of domain structure on protein folding and function.
In eukaryotes there is frequently a correlation between exons in DNA (genes) and domains in protein structure.This correlation potentially allows the joining of exons in new combinations to give rise to new proteins from semi-functional domains to give new multidomain proteins with a good likelyhood of functioning (e.g. could combine a domain for binding a sugar with a domain for binding ATP to give a new sugar phosphatase).
Large proteins are generally composed of a number of independently folded regions known as domains. This allows the rapid independent folding of these regions which can then come together to give the finished protein. Frequently active sites are split between two or more domains with differing binding specificities or activities.
(8) 3. Circle (in red for key) the best answer for each of the following:
a. Glycolytic enzymes are synthesized:
in the cytosol, on the endoplasmic reticulum, on the
rough endoplasmic reticulum, in the golgi complex.
b. Of the following amino acids,those
which are most likely to be found in a proteins interior are:
(ala, ser, & cys), (gly,
leu, & pro), (tyr, glu,&
ala), (his, arg, & gly).
c. The most important force or
bond in causing a protein to fold up into a compact form is: hydrogen
bonding, disulfide bonding, hydrophobic
forces, van der Waals forces.
d. The most important/characteristic
"bond" type for maintaining secondary structures in
protein assemblies is: hydrophobic, van der Waals, hydrogen,
covalent, ion pair.
(9) 4. Briefly discuss the roles of disulfide bonds (if any) in each of the following: protein folding, protein structure, and protein function.
a) protein folding - disulfide bonds are generally not important in the folding process.
b) protein structure- disulfide bonds are important for maintaining a proteins 3-D structuire in "harsh" environments outside of the cell (e.g. blood plasma, tears, digestive tract). The have little or no function in intracellular proteins.
c) protein function- disulfide bonds are needed to maintain a protein's structure, and thus its function, in environments outside of the cell.
(14) 5. On the axis below draw typical kinetic curves for an allosteric enzyme such as we discussed in class under the following conditions: a) with no effectors present, b) in the presence of a positive effector, and c) in the presence of a negative effector. Label them!
d. Which of these curves shows the least cooperatively? Explain briefly why this should be so.
The (+) effector curve is the least cooperative. If an effector "fully turns on" a protein it increases the affinity to its maximum value - therefore there can't be any cooperativity (its already binding as well as possible).
e. Challenge question: What would the effect of a competitive inhibitor on this enzyme if it were present at less than half saturating concentration? As your answer you can draw the curve for this situation on the plot above - label it!
Note that the competitive inhibitor initially acts as an activator since it binds in a substrate site, allosterically "turning on" the enzyme at low [S]. however, at high [S] it occupies substrate sites, resulting in a true inhibition, until the [S] finally overcomes it at higher concentrations.
(12) 6. List and describe as briefly as possible the six levels of protein structure we have discussed in class.
Primary (1°)- the sequence order of aa residues in the peptide linked by peptide bonds.
Secoundary (2°)- the steric relations of residues nearby in the primary structure. Due to peptide backbone interactions and stabilized by H-bonds between peptide groups. Often characterized by repetitive "helical" arrangements such as a-helices and b-strands.
Tertiary (3°)- the steric relations of residues distant in the primary sequence. Often refered to as the overall folding pattern of the protein. Due to interactions between residue side chains. For soluble proteins hydrophobic forces are th primary drive for tertiary folding.
Super Secondary (Motifs)- the steric relations (associations) of secondary structural elements (e.g. aa, bab, b-barrel, etc.)
Domain- independent folding regions of a peptide or within a tertiary structure.
Quaternary- the steric relationships between two or more tertiary structures.
(8) 7. A typical plot for enzyme activity as a function of temperature is shown below. Describe/explain the phenomena underlying this curve.
(12) 8. Consider the process of protein folding and Briefly answer the following questions:
a. Why is it often said that proteins do not achieve the folded state with the lowest global free energy?
It would take too long to explore all of the possible folded states in order to find the one with the lowest DG.
b. Why are alpha-helices so commonin globular proteins?
They fold faster than other structures because of
- rapid nucleation and
- extremely rapid bi-directional (cooperative) propagation.
c. What advantage is there to breaking a large protein up into domains?
Smaller domains can fold faster than large proteins. Many small domains can also be folding simultaneously (in parallel). The resulting folded domains can then assemble to 3° structures providing a major increase in the overall folding rate.
d. How do chaperones aid proteins in achieving their native folded structures?
They keep the peptide chain from folding too rapidly and prematurely, preventing the formation of non-functional proteins.
Last modified 5 March 2004