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NAME _______________________________

Exam II I. _________________/30
October 16, 2017
Biochemistry I II. _________________/45
BI/CH 421/621
III. _________________/25

TOTAL ________________/100

I. MULTIPLE CHOICE. (30 points; 3 pts each)

Choose the BEST answer to the question by circling the appropriate letter.

1. A D-amino acid would interrupt a b-strand made of L-amino acids. Another

naturally occurring constraint on the formation of a b-strand is the presence
of:

A. a negatively charged Arg residue.

B. a positively charged Lys residue.
C. two Gly residues side by side.
D. a nonpolar residue near the carboxyl terminus.
E. a Pro residue.

2. Which of the following is not considered one of the four major levels of
protein structure?

A. quaternary structure
B. primary amino acid sequence
C. domain structure
D. three-dimensional conformation of entire polypeptide
E. secondary structure

3. Which statement about 3° structure determination is false ?

A. In X-ray crystallography, a structure determined at 2 Å resolution

has more detail than one determined at 5 Å resolution.
B. In NMR, COSY is able to determine bond angles.
C. Some of the structure could be conferred by crystal packing.
D. NMR structures are generally at higher resolution than X-ray
structures, yet they reveal details of conformational flexibility.
E. NMR can readily distinguish between Thr and Val, whereas X-ray
crystallography cannot.

4. The concept of "induced fit" refers to the fact that:

A. when a substrate binds to an enzyme, the enzyme induces a loss of

water (desolvation) from the substrate.
B. substrate binding may induce a conformational change in the enzyme,
which then brings catalytic groups into proper orientation.
C. enzyme-substrate binding induces an increase in the reaction
entropy, thereby catalyzing the reaction.
D. enzyme specificity is induced by enzyme-substrate binding.
E. enzyme-substrate binding induces changes in the substrate towards
the transition state.
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NAME _______________________________

5. The major driving forces involved with the formation of very large multi-
subunit protein complexes, for example the protein capsid of a virus, are:

A. free energies of hydrolysis of such molecules as ATP and GTP.

B. the same weak, noncovalent interactions that determine the native
conformation of a single polypeptide.
C. the free energies of binding by small ligands.
D. entropy increases resulting from the formation of many new peptide
bonds.
E. entropy decreases resulting from the formation of symmetric
crystalline shapes, for example, icosahedrons.

6. Which of the following statements is false ?

A. Each strand of the protein chain in collagen is a left-handed

helix.
B. For S Æ P, an enzymne catalyst will always increase the reaction
rate.
C. At the end of an enzyme-catalyzed reaction, the functional enzyme
becomes available to catalyze the reaction again.
D. Substrate binds to an enzyme's active site.
E. Collagen is an abundant globular protein in mammalian organisms.

7. For the study of a protein in detail, an effort is usually made to first:

A. conjugate the protein to a known molecule.

B. determine its amino acid composition.
C. determine its molecular weight.
D. determine its amino acid sequence.
E. purify the protein.

8. In an a helix, the R groups on the amino acid residues:

A. are found on the outside of the helix spiral.

B. generate the hydrogen bonds that form the helix.
C. stack within the interior of the helix.
D. cause only right-handed helices to form.
E. alternate between the outside and the inside of the helix.

9. Amino acid residues commonly found in type I and/or type II b-turns are:

A. Ala and Gly.

B. Pro and Gly.
C. two Cys.
D. hydrophobic.
E. those with ionized R groups.

10. Pauling and Corey's studies of the peptide bond showed that:

A. for a protein in solution at pH 7, any one of a large number of

conformations is equally probable.
B. the primary structure of all proteins is very similar, although the
secondary and tertiary structure may differ greatly.
C. the peptide bonds in proteins are very unusual, bearing almost no
resemblance to peptide bonds in small model compounds.
D. the peptide bond is essentially planar, with no rotation about the
C-N axis of the amide bond.
E. the structure of a peptide bond is so complex that even Pauling
could not understand it.
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NAME _______________________________

II. SHORT ANSWER. (45 points)

Give a brief answer or diagram for each problem or question below.

11. In the lab, when you use BSA for making a standard curve for a colorimetric
assay, what substance are you trying to measure? What quantity is this assay
meant to determine? What assumptions are you making relative to an unknown
(not BSA) you are measuring using that standard curve? (3 pts)

12. What was the purpose of heating the BSA prior to digestion with trypsin in
the Experiment in Chapter 2 of the laboratory? Would not heating have
affected the results? How? (2 pts)

13. In lab, you used histidine with its 3 dissociable protons: one with a pK a of
2.3, one with a pK a of 6.5, and the third with a pK a of 9.7. Sketch a
properly labeled titration curve for histidine (HHis+) titrated with NaOH;
indicate where on the curves the pH = pK a , and indicate the region(s) of the
curve in which the highest buffering occurs. (3 pts)

14. Diagram a peptide bond from one Ca to the next (no R-groups please) in the
resonance form that best explains its characteristics. For the Ca -Cpeptide and
the Npeptide-Ca bonds, label each by the name of the angle that describes its
conformation. (3 pts)
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NAME _______________________________

15. A biochemist purified a new enzyme and subjected a sample to SDS-PAGE (SDS-
polyacrylamide gel electrophoresis). The results showed that there were three
different proteins present with relative molecular weights of 20 kDa, 25 kDa,
and 40 kDa. When sedimentation equilibrium ultracentrifugation was performed
on the preparation, a molecular weight of 130 kDa was determined. What is
the quaternary structure of this enzyme? Use proper nomenclature. Diagram
the expected intensities of subunits on a stained SDS-PAGel. (5 pts)

16. The X-ray crystallographic analysis of proteins often fails to reveal the
positions of parts of the peptide chain, particularly the N- and C-terminal
few amino acids. Explain. (2 pts)

17. Describe the primary, secondary/tertiary, and quaternary structure of

collagen. How is pro-collagen different from collagen? (5 pts)

18. In the process called ________, phenylisothiocyanate (below) reacts with N-

terminal amino group of a polypeptide, ultimately cleaving the first peptide
bond releasing a phenylthiohydantoin derivative of the N-terminal amino acid.
(2 pts)

19. How would the following agents or procedures interfere with or disrupt the
different levels of protein architecture? Why? (3 pts)
a) Addition of SDS

b) Oxidation of cysteine with performic acid to cleave disulfide bonds

c) Addition of proteases such as trypsin

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NAME _______________________________

20. You purified a 20 kDa protein and determined its mass spectrum by ESI-MS and
got the data shown below. Circle the letters for the following statements
about the data if they are true? (the numbers above each peak are the m/z for
that peak) (3 pts)

a. you must have more than one protein in the sample because there is more
than one peak in the spectrum.
b. the peaks with a higher m/z are more charged than those with the lower
m/z.
c. the peak at m/z = 1001.0 has a z = +20
d. the protein has a mass of 1001.0 Da, not 20 kDa as you had first thought.
e. you cannot interpret this mass spectrum without knowning the amino acid
composition.

21. Several biotechnology/pharmaceutical companies have explored the idea of

using "stapled" peptides for stabilizing small therapeutic peptides as a-
helices. Unnatural amino acids with reactive side-chains are introduced into
these peptides and then induced to covalenlty cross-link to form the
"moleculaar staple," which theoretically stabilizes the a-helical
conformation of the peptide (see below).

a. For the amino acids X and Y to form the cross-link shown above, how far
apart in the primary structure of the peptide must they be? Briefly, explain
your answer. (2 pts)

b. Before and after the stapling reaction, the circular dichroism spectra
were determined (see below). Which spectra belongs to the unstapled and which
belongs to the stapled peptide, and why. Was this idea successful? (2 pts)
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NAME _______________________________

22. Your Edman Degradation sequenator was broken and there is a postal strike so
you could not send your peptide for sequence determination. You decide to use
chemical techniques. This decapeptide had an amino acid composition: (Gly)3,
(Phe)2, Pro, Arg, Met, Leu, Ala.
A. 1) Reacting the native peptide with FDNB followed by complete hydrolysis
(N-terminal analysis) released 2,4-dinitrophenylglycine.
2) Incubation with CNBr yielded two fragments, a tetrapeptide with
composition (Gly)2, homoserine lactone, and Phe, and a hexapeptide. The
tetrapeptide yielded 2,4-dinitrophenylglycine, and the hexapeptide yielded
2,4-dinitrophenylleucine after N-terminal analysis with FDNB.
3) Proteolytic cleavage of the native decapeptide by trypsin gave two
fragments, a hexapeptide and a tetrapeptide; N-terminal analysis of the
tetrapeptide yielded 2,4-dinitrophenyl-phenylalanine.
4) Chymotrypsin cleavage of the native peptide gave a tripeptide composed
only of glycine and Phe, and a heptapeptide.
B. The information in A) should yield the sequence of all but the C-terminal
residues. You decide to use your brand new tandem MS/MS instrument. You
perform MS/MS on the C-terminal tryptic tetrapeptide to determine the
sequence of this remaining peptide. The pattern gives you MW fragments of
444, 369, 280, 279, 165, 164, 75.
[NOTE: MW of tetrapeptide is 444 and MW of Ala, Gly, Phe, and Pro are 89,
75, 165, and 115, respectively. Phe-Pro has MW of 280.]
Put the sequence based on this information in the spaces below. (10 pts)

____ ____ ____ ____ ____ ____ ____ ____ ____ ____

III. MATCHING. (25 points)

23. Match the types of protein structure on the right with techniques for
structure determination for which they can be determined effectively. In the
blank to the right of number 1, cite an example of a type of structure that
corresponds to your answer. (5 pts)

________1. Circular Dichroism _____________________ A. primary structure

________2. Diagonal Peptide Mapping B. tertiary structure
________3. Edman Degradation C. secondary structure
________4. Gel filtration & SDS-PAGE D. quaternary structure
E. disulfide bond structure

24. For each EC enzyme category or protein sequence determination method on the left, match to
the most appropriate enzyme with a trivial name or description of the method on the right. (8
pts)
_________1. oxidoreductase A. Tyrosine transaminase
_________2. transferase B. determination of disulfide bond formation
_________3. hydrolase C. determination of amino acid content
_________4. isomerase D. trypsin
_________5. ligase E. Tyrosine-tRNA aminoacyl synthetase
_________6. amino acid analysis F. blocking disulfide bond formation
_________7. diagonal peptide mapping G. lactate dehydrogenase
_________8. reduction and alkylation H. phosphoglucomutase
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NAME _______________________________

25. Match the items on the left with the BEST item for each on the right: (5 pts)
_________1. Trivial enzyme A. Kd
name (only)
_________2. Systematic enzyme B. k cat
name and EC
number
A B P Q
_________3. sequential random C.
bi bi E E

A B P Q
_________4. Enzyme D.
proficiency E FAB
E

B A Q P

A P B Q
_________5. Enzyme turnover E.
rate E E

F. k cat/k uncat
G. kinase
H. isomerase
I. EC 1.2.1.32 a lyase
J. EC 1.2.1.32 an oxidoreductase

26. From the types of bonds and interactions on the right, identify which is most
responsible for the structures described on the left. If there are two types
of bonds of nearly equal responsibility, identify both. There are no more
than two for each. (7 points)
_________1. a tight fit (≤2Å) of complementary shapes A. ionic interactions
of a ligand to its receptor
_________2. secondary structure of proteins B. hydrophobic interactions
_________3. the triple helix of collagen C. van der Waals interactions
_________4. the compactness of the interior of D. covalent bonds
myoglobin
_________5. the association of collagen molecules to E. hydrogen bonds
make collogen fibrils
_________6. the association of hemoglobin subunits in
its quaternary structure
 Answer Key for Exam 2 10/16/2017 Page 1
No. on
Test Correct Answer
1 E
2 C
3 D
4 B
5 B
6 E
7 E
8 A
9 B
10 D
11
You are measuring PROTEIN. You are quantifying protein concentration or
amount of protein. You assume that BSA reacts to give a color in a similar
way as what you are measuring.
12
1. Heating denatured the BSA, thus making all the Arg and Lys as accessible
to trypsin as possible.

2. Without heat the number of these residues measured would likely have been
fewer due to some being buried or at least the reaction taking longer to
reach completion.

--
13
The flatest part the pH = pK a and are the best buffering portions of the
curve.

9.7
pH

6.5

2.3

1 2 3
OH- Equivalents

--
14
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No. on
Test Correct Answer
15 Since there must be at least one of each subunit, the MW would be ≥85 kDa.
Sedimentation equilibium ultracentrifugation measured the native MW of 130
kDa, therefore the only combination that gives 130 would be 45 kDa more, so
there must be two each of the smaller subuinnts (20x2 + 25x2 + 40 = 130). So
the quaternary structure would be ab2 g 2 , where either the 20 or 25 kDa
subunits are b or g and the 40 kDa subunit is a. Make sure each subunit is
defined.
There will be three bands 20 kDa will be about equal intensity with the 40
kDa due to half the size, but double the number with the 25 kDa with slightly
more intensity than the 20 kDa.

16

In a protein crystal, the residues at the end of a polypeptide chain, and

some interior residues, may experience fewer intramolecular contacts or be
innately mobile. They tend to not occupy a set position in the crystal (less
ordered, more mobile). This disorder prevents them from generating a
coherent diffraction pattern when the x-ray beam hits these parts of the
protein and without a diffraction pattern the electron density cannot be
generated.
17
Collagen's primary structure is its amino acid sequence, which is a
repeating triplet of G-P-X (X can be Pro or HydroxyPro among others).
Secondary structure is the left-handed helical conformation.
Tertiary structure is essentially the same as its secondary structure as it
is a fibrous repetitive protein.
Quaternary structure is the association of subunits and and the arrangment of
three chains in a right-handed triple helix (coiled coil); a3.
Procollagen has globular domains at the N- and C-termini, which direct the
coiling of the three strands.
18
Edman Degradation
19
a) SDS is a detergent would interfer with the hydrophobic interactions and
perturb secondary, tertiary, and quaternary structures, but not the primary.
b) If a protein had disulfide bonds, these would be broken. This might be
considered disruption of primary (covalent) structure, but it might also
affect tertiary and quaternary structure, maybe some secondary.
c) Trypsin will catalyze the hydrolysis of peptide bonds. This will affect
the primary structure. As such, all other structures may potentially be
affected (secondary, tertiary, and quaternary structure).
20
Only circle "c"
21
a. there are 2 turns of the helix in the diagram, so that would be 2 x 3.6
AA/turn = 7 AA.
b. "A" belongs to the stabled -helical spectrum (minima at210 & 222 nm)
"B" belongs to the un-stapled peptide as a coil with minima at 200 nm.
22
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No. on
Test Correct Answer
22 The amino-terminal residue is Gly. Cyanogen bromide cleavage gave the
following:
Gly-(Phe, Gly)-Met/Leu-(Phe, Arg, Gly, Ala, Pro). Trypsin cleaves on the
carboxyl side of Arg, so after cleavage by trypsin, there is:
Gly-(Phe, Gly)-Met-Leu-Arg/Phe-(Gly, Ala, Pro). Chymotrypsin hydrolyzes the
amino side of Phe, which gives the N-terminal tripeptide as a
glycylglycylphenylalanine tripeptide. The other expected cleavage at the
second Phe is precluded by an adjacent Pro (it can't be at the C-term as the
FDNB gave Phe at the N-term of the tryptic tetrapeptide). Therefore:
Gly-Gly-Phe-Met-Leu-Arg-Phe-Pro-(Ala, Gly) The sequence of the
carboxyl-terminal amino acids cannot be resolved without additional
information from MS/MS. The tryptic tetrapeptide is either FPAG or FPGA.
The MW data from the MS/MS data shows evidence for release of a terminal Gly
(MW=75) rather than a terminal Ala (MW=89). Also, there is evidence of a FPA
fragment (MW=369) rather than a FPG (MW=355). Therefore, the final sequence
is:

Gly-Gly-Phe-Met-Leu-Arg-Phe-Pro-Ala-Gly
--
23
1. C (a-helix or b-sheet)
2. E
3. A
4. D
24
1. G
2. A
3. D
4. H
5. E
6. C
7. B
8. F

25
1. G
2. J
3. D
4. F
5. B

26
1. C
2. E
3. E and C
4. B (not wrong is C or E included as second)
5. D
6. B (not wrong is C or E included as second)