CHEM151/251

Biochemistry I
Homework
Chapter 4: The Three-Dimensional Structure of Proteins

Chapter 4: 1, 3-4, 7-10, 12, 13

1. In x-ray studies of crystalline peptides, Linus Pauling and Robert Corey found that the C-N bond
in the peptide link is intermediate in length (1.32 ) between a typical C-N single bond (1.49 )
and a C=N double bond (1.27 ). They also found that the peptide bond is planar (all four atoms
attached to the C-N group are located in the same plane) and that the two -carbon atoms
attached to the C-N are always trans to each other (on opposite sides of the peptide bond):

a. What does the length of the C-N bond in the peptide linkage indicate about its strength
and its bond order (i.e., whether it is single, double, or triple)?
Peptide bond has resonance due to the large dipole moment between the carboxyl and amino
groups. This resonance makes the molecule stronger and more rigid and is thus stronger than
the typical C-N bond. Bond order is directly linked to bond length. It can thus be inferred that
C==N is the strongest and is of the highest order and the C-N bond is the weakest with the
lowest order. The C-N in the peptide bond is the intermediately strong bond and hence lays
between.

b. What do the observations of Pauling and Corey tell us about the ease of rotation about the
C-N peptide bond?
The planarity of the peptide bond is indicative of the fact that the C and N terminals of
the molecule are 180 degrees to each other, are virtually fixed and assume a trans-
configuration. This configuration makes it nearly impossible for rotation to occur at the
peptide bond.

3. Hair grows at a rate of 15 to 20 cm/yr. All this growth is concentrated at the base of the hair fiber,
where -keratin filaments are synthesized inside living epidermal cells and assembled into
ropelike structures. The fundamental structural element of -keratin is the helix, which has 3.6
amino acid residues per turn and a rise of 5.4 per turn. Assuming that the biosynthesis of -
helical keratin chains is the rate-limiting factor in the growth of hair, calculate the rate at which
peptide bonds of -keratin chains must be synthesized (peptide bonds per second) to account for
the observed yearly growth of hair.

5.4Ang/3.6AA= 1.5Ang per amino acid/ per turn

1 Angstrom =1*10^-10m
Using 20cm of hair:
20/ (365)(24)(60)(60)=6.3* 10^-7cm/s
Rate at which amino acids are added: 6.3*10^-9/1.5*10^-10= 42AA/s and thus 42 peptide bonds
per second.
4. The unfolding of the -helix of a polypeptide to a randomly coiled conformation is accompanied
by a large decrease in a property called its specific rotation, a measure of a solutions capacity to
rotate plane-polarized light. Polyglutamate, a polypeptide made up of only L-Glu residues, has the
_-helical conformation at pH 3. When the pH is raised to 7, there is a large decrease in the specific
rotation of the solution. Similarly, polylysine (L-Lys residues) is an -helix at pH 10, but when the
pH is lowered to 7 the specific rotation also decreases, as shown by the following graph.
 What is the explanation for the effect of the pH
changes on the conformations of poly(Glu) and
poly(Lys)? Why does the transition occur over such
a narrow range of pH?

Poly-glutamate has an helical conformation at pH

3. When the pH is raised to 7, there is a large
decrease in the specific rotation of the solution due
to the carboxyl groups of poly (Glu) being fully
deprotonated. This results in the unfolding of the
helix. Similarly, poly-lysine, an helix at pH 10,
can unfold due to the protonation of the amino
groups of poly (Lys). A repulsion is formed among
these positively charged groups. This narrow transition occurs because deprotonation of only a
few of each of the amino acids donate charge to the helix. This small charge can lead to repulsion
and evidently rapid unfolding of the helix.

7. Our growing understanding of how proteins fold

allows researchers to make predictions about
protein structure based on primary amino acid
sequence data.
a. In the amino acid sequence above, where
would you predict that bends or turns
would occur?
Bends and turns have the highest possibility
of forming at Pro residues 7 & 19. Bends or turns are may also occur at the Thr residue
(residue 4) or the Ile residue (residue 1).

b. Where might intrachain disulfide cross-linkages be formed?

Formed between Cys residues 13 and 24.

c. Assuming that this sequence is part of a larger globular protein, indicate the probable
location (the external surface or interior of the protein) of the following amino acid
residues: Asp, Ile, Thr, Ala, Gln, Lys. Explain your reasoning.

Amino acids with ionic (charged) or strongly polar neutral groups (Asp, Gln, and Lys) would be located
on the external surface, where they interact with water. Residues with nonpolar side chains (such as Ala
and Ile) are hydrophobic and would be located in the interior of the cell. Thr is of intermediate polarity
and could be found either in the interior or on the exterior surface of the protein.

8. Under the proper environmental conditions, the salt-loving bacterium Halobacterium halobium
synthesizes a membrane protein (Mr 26,000) known as bacteriorhodopsin, which is purple
because it contains retinal. Molecules of this protein aggregate into purple patches in the cell
membrane. Bacteriorhodopsin acts as a light-activated proton pump that provides energy for cell
functions. X-ray analysis of this protein reveals that it consists of seven parallel -helical
segments, each of which traverses the bacterial cell membrane (thickness 45 ). Calculate the
minimum number of amino acid residues necessary for one segment of -helix to traverse the
 membrane completely. Estimate the fraction of the bacteriorhodopsin protein that is involved in
membrane-spanning helices. (Use an average amino acid residue weight of 110.)

Using the parameters from Problem 3 (3.6 AA/turn, 5.4 /turn), there are 0.67 AA/ along the
axis of a helix. Thus, a helix of length 45 requires a minimum of (45 ) (0.67 AA/) 30
amino acid residues. The membrane protein has an Mr of 26,000 and average AA M r of 110.
Thus the protein contains 26,000/110 240 AA. Of these, (30 AA/helix)(7 helices) 210 AA are
involved in membrane-spanning helices, which is 210/240 0.87, or 87%, of the protein

9. Is myoglobin a motif, a domain, or a complete three-dimensional structure?

Myoglobin is a whole protein as a single polypeptide, so it is a complete 3-dimensional structure

possessing a domain and motifs. No quaternary structure.

10. Examine the two proteins labeled (a) and (b) below. Which of the two Ramachandran plots,
labeled (c) and (d), is more likely to be derived from which protein? Why?
From the observation of the Ramachandran plots, one can clearly see an accumulation of hits in the upper

left corner of the plot, which is indicative of the presence of beta-pleated sheets. Since b protein possesses
no sheets, it can be inferred that the first Ramchandran plot belongs to a protein and the second belongs to
the b protein.

12. A sample (660 mg) of an oligomeric protein of Mr 132,000 was treated with an excess of 1-
fluoro-2,4-dinitrobenzene (Sangers reagent) under slightly alkaline conditions until the chemical
reaction was complete. The peptide bonds of the protein were then completely hydrolyzed by
heating it with concentrated HCl. The hydrolysate was found to contain 5.5 mg of the following
compound:

2,4-Dinitrophenyl derivatives of the -amino

groups of other amino acids could not be found.
 a. Explain how this information can be used to determine the number of polypeptide chains
in an oligomeric protein.

Only one peptide was located. This means that this peptide is the only one located at the
amino acid terminus. The number of moles of this yielded protein to the number of moles
of protein gives the number of polypeptide chains.

b. Calculate the number of polypeptide chains in this protein.

Number of polypeptides in a large protein can be determined by measuring the amount of N-terminal
amino acid(s).
In this case, using FDNB to label the N-terminal amino acids, it produced only one DNP-amino acid:
DNP-val (protein sample => 5.5 mg DNP-val).
a. All we need to do is find the moles of DNP-val formed from the number of moles of the protein
(132,000 daltons) in a 660 mg sample.
b. Lets do it ! First moles of protein = (0.66 g protein) / (132,000 g/mole) = 5 x10-6 moles of protein.
Now, moles of DNP-valine (MW DNP-val = 283 daltons).
Thus the moles of DNP-val = 0.0055 g / (283 g/mole) = 1.9 x 10-5 moles.
So then, 1.9 x 10-5 moles DNP-val / 5 x 10-6 moles protein = 3.9 or 4 N-terminals per protein.
Thus, there are 4 polypeptides that make up this protein.
c. What other protein analysis technique could you employ to determine whether the
polypeptide chains in this protein are similar or different?

The simplest would be do run an SDS- PAGE gel with molecular weight standards. It is
likely that they all come out to be 33,000 daltons (33 kD) if they were all identical
forming only one band. But, it could be that they came from different cistrons, and in
that case there would likely be different MW bands even though they all had valine as the
N-terminal amino acid.

13. Which of the following peptides is more likely to take up an a-helical structure, and why?
a. LKAENDEAARAMSEA
b. CRAGGFPWDQPGTSN
A is more likely to take up an alpha helical structure due to the presence of so many helix formers A and
L. B possesses five helix breakers that will make this configuration unfavorable.