Biochemistry 461 Name: ______________________________

Nov. 3, 2011
Prof. Julin
Exam II
(100 points)

NOTE:
read the questions carefully,
think about your answers, and,
be sure that you answer all parts of the questions.
Provide specific information that answers the question being asked.
Always show your work or explain your reasoning.

You may use a calculator, but only for computation. Any other use is a violation of the
University’s Code of Academic Integrity.
No other electronic equipment may be used - that is, turn off your cell phone!

Honor Pledge
Please write the following sentence in the box, and then sign your name:

“I pledge on my honor that I have not given or received any unauthorized assistance
on this examination.”

Some equations and formulas which might (or might not) be useful:

a) )GN° = !RT ln(Keq)Keq = [products] / [reactants]

)GN° = +RT ln(Kd) Kd = [reactants] / [products]

b) )G = )H ! T)S

c) )G = )GN° + RT ln {[products]/[reactants]}

d) R = 0.0083 kJ/K/mol e) T (K) = T (°C) + 273

f) 2 = [PCL]/[P]T = [L]T / (Kd + [L]T)

g) Hill equation: log [2 / (1 ! 2)] = n log([L]T) ! log (Kd)

1. (10 points) i) (6 pts) The two bonds indicated by arrows in the figure below are single bonds,
yet their possible rotation angles and conformations are limited. Explain the factors that limit the
possible conformations of each bond.

bond 1:
1
H R O
N C C
C H N C
O H

bond 2:

ii) (4 pts) Suppose you want to determine the value of the angle Phi (N) in the peptide
shown below. Draw arrows to the two bonds in the figure that you would look at to figure out the
value of Phi.

H R O
N C C
C H N C
O H

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2. (10 points) A common experimental approach to studying proteins is site-directed
mutagenesis. You change one specific amino acid residue in the protein to a different residue
(e.g., you might change Asp94 in hemoglobin to Ala), and then tests to see whether that specific
change affects the function of the protein.
One concern is always that the amino acid change might alter or destabilize the three-
dimensional structure of the protein, and thereby affect the function indirectly. It is often difficult
to be certain that the mutation has not changed the structure, but you can reduce the chances of
that by being careful as to which amino acid you choose to insert by mutation.
There are two amino acids that you would not insert, if you are smart: Glycine and
Proline. Explain why not, in terms of the unique structural properties of these two amino acids.

glycine

proline

3. (12 points) i) Describe briefly the non-covalent interactions that stabilize a two-stranded beta-
sheet, and the chemical groups on the beta-strands that are involved.

ii) Now consider the “steric zipper” structure that has been proposed for amyloid fibers
composed of the A$ peptide. What additional non-covalent interactions are involved in
stabilizing this structure. What specific groups on the peptide are involved?

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4. (12 points) i) RNaseA was denatured in the historic Anfinsen experiment by adding both urea
and beta-mercaptoethanol. How did each of these substances contribute to the denaturation of the
protein?

ii) Suppose the RNaseA were denatured by the addition of urea alone, without beta-
mercaptoethanol. Call the denatured protein produced in this way U’. Now suppose the urea is
removed by dialysis, which allows the RNaseA to renature (producing N).
For the following equilibrium:

–urea
U’ N
+urea

how would the entropy change for folding ()Sf , for U’ ÷ N) of RNaseA that had been
denatured in this way compare to the )Sf for RNaseA denatured with both urea and beta-
mercaptoethanol, as in the real Anfinsen experiment described in part (i)? Explain.

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5. (14 points) i) (4 pts) Short peptides that consist entirely of alanine residues will fold to form
relatively stable alpha-helices in aqueous solution.
The figure below shows a melting curve for a small peptide consisting of 18 alanine
residues (Ala18). Draw an arrow to the part of the curve where the peptide is mostly folded.
Estimate the melting temperature (Tm) for the peptide from this curve. Show your work.

ii) (10 pts) Described on the next page are two experiments in which one or two amino
acid residues in the peptide were changed. The stability of the new peptide was measured by
repeating the melting experiment illustrated in the graph, above.
For each case, use the axes that are given to draw the new melting curve that would be
obtained if the melting experiment were repeated, but with the peptides described in each part.
Explain why you drew the new curve(s) as you did, and explain specifically why and
how the amino acid change would affect the stability of the alpha helix.

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Question 5, part ii, continued a) A peptide with Glu at residue # 3 compared to one with
Glu at residue # 16. You must draw two curves, and label which curve goes with which peptide
(and explain).

2 4 6 8 10 12 14 16 18
– Ala - Ala - Glu - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala
compared to:
2 4 6 8 10 12 14 16 18
– Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Glu - Ala - Ala

b) The Ala18 peptide compared to one with Glu at residues #7 and #10.

2 4 6 8 10 12 14 16 18
– Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala
compared to:
2 4 6 8 10 12 14 16 18
– Ala - Ala - Ala - Ala - Ala - Ala - Glu - Ala - Ala - Glu - Ala - Ala - Ala - Ala - Ala - Ala - Ala - Ala

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6. (10 points) Suppose you have a protein P that binds to sugars:

P + sugar W PCsugar

You measure binding of the protein to three different sugars by calorimetry, which gives you
thermodynamic quantities for the binding reaction written above. The standard free energy
changes for the binding reactions are given in the Table, below.

sugar )G°N (kJ/mol)

galactose !10
fructose !6
mannose !8

Use the axes given below to draw the three binding curves (2 vs. [sugar]) that you would get for
the three sugars binding to the protein. Add numerical labels to the y-axis. Label clearly which
curve corresponds to which sugar, and explain why you drew the curves as you did. (NOTE -
you can answer this question qualitatively. You need not do any calculations (although you can
if you want to).)

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7. (8 points) Given below are binding curves for a protein P that binds to some ligand L. The
experiment was done with [L]t >> [P]t.
i) Suppose you repeat the experiment, but with a protein concentration that is 2-times
greater than was used in the experiment shown. Draw the new curves that you would get, in each
case. Explain why you drew the curves as you did.

ii) Suppose you repeat the original experiment but with a mutant protein that has 2-times
lower affinity for the ligand. Draw the new curves that you would get, in each case. (The total
protein concentration ([P]t) is the same as for the printed curves in the figure.) Explain why you
drew the curves as you did.

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8. (12 points) Hemoglobin has four subunits, but it can be thought of as a dimer of dimers:
("1$1)C("2$2). In fact, the tetramer can dissociate into two dimers:

("1$1)C("2$2) W ("1$1) + ("2$2)

i) The individual dimers ("1$1) and ("2$2) can both bind oxygen. The graph given below
shows the oxygen binding curve for the normal hemoglobin tetramer. Add to the graph the plot
you would get for one of the dimer forms (say, ("1$1)). Explain why you have drawn the new
curve the way that you did. What is the shape of your new curve?

ii) Consider the following equilibrium and the equilibrium constant for the reaction.

Keq
("1$1)C("2$2) W ("1$1) + ("2$2) Keq = ["1$1] ["2$2] / [("1$1)C("2$2)]

The value of Keq for hemoglobin is Keq = 3 × 10!10 under physiological conditions.

Suppose that the equilibrium constant were measured again, but this time in the presence
of 2,3-BPG. How would you expect the magnitude of the equilibrium constant to compare to that
in the absence of 2,3-BPG (greater than, equal to, or less than)? Explain how you arrived at your
answer.

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9. (12 points) Consider the hypothetical completely cooperative mechanism as it would apply to
oxygen binding to hemoglobin:

Hb + 4O2 W HbC(O2)4

i) Shown below is the Hill plot for O2 binding to hemoglobin (taken from a figure in the
textbook, with hypothetical experimental data points added). Explain why this Hill plot for
hemoglobin proves that O2 binding to hemoglobin does not follow the completely cooperative
mechanism written above.

ii) The figure to the right shows the concerted mechanism

as it applies to hemoglobin. (Note that the equilibrium arrows
were all drawn the same size for convenience, and you should not
worry about their relative sizes in answering the question.)
In the concerted mechanism on the right, draw a circle
+O2 +O2
around the two species that would always be present at very low
concentration, regardless of the pO2. Explain briefly why you
chose those species. O2 O2

+O2 +O2

O2 O2 O2 O2

+O2 +O2

O2 O2 O2 O2
O2 O2

+O2 +O2

O2 O2 O2 O2
O2 O2 O2 O2
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