UNIVERSITY OF TORONTO

FACULTY OF ARTS AND SCIENCE

BCH210HF - BIOCHEMISTRY I

Term Test 1: October 15, 2015

Total Duration: 2 hours
Examiner: Dr. R. Reithmeier

NO AIDS ALLOWED

This examination is worth 35% of the final grade for the course. There are
35 multiple-choice questions worth 1 mark each for a total of 35 marks. You
are to choose ONE answer for each question and fill in the appropriate circle
on the computer card with a soft lead pencil (not pen!). Marks are not
deducted for wrong choices but no marks are given if more than one circle is
filled in per question.

It is NOT a good idea to leave the entering of your answers on the computer
card until the last few minutes of the exam. We cannot help you if you make
transcriptional errors. Please allow sufficient time to enter your answers on the
Scantron card. Responses on this exam question paper will not be graded.

When you are not entering answers on your card, please cover the answer
card with your question paper. You are responsible for maintaining
academic integrity in this course and will be held accountable for the
actions of others if implicated.

Please note that the examiners make every effort to ensure there is only one
suitable answer for each multiple-choice question. However, if you are
convinced that a particular question has two answers, you should select the
answer that seems MOST appropriate. Do NOT skip the question. Invigilators
cannot interpret questions or provide any assistance with the material.

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Multiple-Choice Questions
Answer ALL 35 multiple-choice questions on the Scantron Card in PENCIL.

Part 1: Choose ONE CORRECT answer:

1. Which ONE of the following statements BEST describes the “take-home” message
from Dr. Reithmeier’s lectures?
a) Mutations in genes can cause amino acid changes in proteins, affecting their function
and resulting in inherited diseases.
b) Proteins take a very long time to fold into their native structures because they must
sample all possible conformational states.
c) The Anfinsen principle that the amino acid sequence defines the final folded state must
be incorrect since the prion protein can assume two very different conformations.
d) Proteins are simple polymers of amino acids joined in a linear fashion through peptide
bonds and can be entirely understood by polymer chemistry.

2. Single amino acid changes in proteins can affect their stability and ability to function
properly. Sickle cell hemoglobin is an example that was discussed in class.
Which ONE of the following statements BEST describes the effect of the Glu6Val
mutation?
a) It causes the hemoglobin tetramer to dissociate into individual subunits.
b) It prevents hemoglobin from binding oxygen.
c) It causes destabilization of a helix and the exposure of a new hydrophobic patch.
d) It causes hemoglobin to unfold, resulting in the loss of the bound iron.

3. Which ONE of the following statements BEST represents the changes observed in the
transition of a peptide from a helical conformation to an unfolded state in urea?
a) The negative ellipticity of the peptide at 222 nm measured by circular dichroism,
would decease in the unfolded state.
b) The Ramachandran plot of the phi/psi angles would not change since the sequence is
the same.
c) The Chou-Fasman helical propensity calculated for the peptide would decrease.
d) The peptide would have the same rise per residue in the two conformations.

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4. We developed our own BCH210H hydrophobicity scale in class sorting the amino
acids from the most hydrophobic to most hydrophilic:
ILVFWYTSPGACHMNQDEKR
Upon inspection of this scale, at least three of the amino acids are clearly in the wrong place.
Which ONE of the following is the BEST amino acid order from most hydrophobic to
most hydrophilic:
a) ILVFWYHPGACTSMNQDEKR
b) ILVFWYTSMCHPGANQDEKR
c) ILVFWYTCSMPGANQDEHKR
d) ILVFWYMPGACTSHNQDEKR

5. Like many serine proteases, Subtilisin has three residues (Asp32, His64, Ser221) that
make up a charge relay network at the active site of the enzyme, plus Asn155 and Met222
that can affect its activity.
Which ONE of the following statements BEST describes the expected results of site-
directed mutagenesis experiments that were carried out on Subtilisin?
a) Replacement of the active site His64 with Lys would result in a fully active enzyme.
b) Replacement of Met222 with Ala would result in a partially active enzyme preventing
oxidation.
c) Replacement of Ser221 with Cys would result in the formation of a disulfide bond
within the protein.
d) Replacement of Asn155 with Asp, which provides a carboxyamide side chain, would
result in a fully active enzyme.

6. The conformation of a peptide often depends on its environment.

Which ONE of the following statements BEST describes the influence of the
environment on synthetic polymers of amino acids?
a) A 20 amino acid polymer of Leu residues would form a water-soluble helix.
b) A 20 amino acid polymer of alternating Leu and Gly would form a stable helix.
c) A 20 amino acid polymer of alternating Leu and Glu would form a helix at pH<2.

d) A 20 amino acid polymer of alternating Leu and Lys would form a -sheet structure
with a basic surface and a hydrophobic surface.

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7. Protein structure is classified into four levels: primary, secondary, tertiary and
quaternary. Which ONE of the following BEST describe the effect of a mutation that
does not cause the protein to misfold but affects the activity of ribonuclease:
a) It would only affect the primary structure.
b) It could be an active-site mutation.
c) It could change the secondary structure, causing a helix to convert to a -strand.
d) It could affect the quaternary structure, causing ribonuclease to dissociate into inactive
monomers.

8. Disulfide bonds are common in secreted and membrane proteins but not in cytosolic
proteins. Which ONE of the following is the BEST reason for this:
a) the cytosol contains high amounts of the reducing agent glutathione.
b) disulfide bonds would destabilize cytosolic proteins.
c) cytosolic proteins lack cysteine residues.
d) cystine is a non-polar amino acid.

9. Imagine that there is a 19-amino acid world rather than the 20 naturally occurring
amino acids used in protein biosynthesis.
Which ONE of the amino acids would not exist and why?
a) Leu because the isomer Ile is also hydrophobic and has similar helix-forming properties.
b) Glu because Asp is also negatively charged and is a strong helix former.
c) Gly because Ala is also a small amino acid and common in reverse turns.
d) Ile because Val is also hydrophobic and a strong beta-sheet former.

10. -barrel proteins found in the outer membrane of E. coli form water-filled channels
that allow polar molecules like sugars to cross the membrane.
Which ONE statement BEST describes the properties of these membrane -barrel
proteins?
aThey contain a hydrophobic interior and a hydrophilic exterior like soluble proteins.
b)They consist of a parallel-sheet where the first strand in the sequence is hydrogen-
bonded to the last strand, forming a closed structure.
c)They consist of transmembrane -strands with alternating non-polar and polar residues.
d) They contain small glycine residues facing the channel that allow for the passage of
large molecules.

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Part 2: For questions 11-20 choose the ONE INCORRECT answer:

11. Which ONE of the following statements concerning the amino acid proline is
INCORRECT?
a) Proline is a poor helix former.
b) Proline is commonly found in reverse turns at the i+1 position.
c) Proline is lacking the amide proton necessary to form a hydrogen bond with the
carbonyl group i-4 residues away.

d) Proline lacks an asymmetric C and therefore is optically inactive.

12. Which ONE of the following statements concerning the right-handed alpha-helix is
INCORRECT?
a) The alpha-helix has hydrogen bonds between NH of residue i and the C=O of residue
i-4.
b) The ideal alpha-helix has phi (φ) and ψ (psi) dihedral angles of about -140° and +135°
respectively, located in the upper left quadrant of a Ramchandran plot.
c) They are found on the surface of globular proteins and project their hydrophobic face
towards the core and their hydrophilic face towards the solvent water.
d) The alpha helix has 3.6 residues per turn, a rise of 1.5 Å per residue, and a rotation of
100 degrees between each successive residue.

13. Which ONE of the following statements concerning the amino acid Tryptophan is
INCORRECT?
a) It has an indole side chain that absorbs UV light at 295 nm.
b) Chymotrypsin specifically cleaves peptide bonds with Trp on the carboxyl side.
c) It is very hydrophobic amino acid resulting in it being fully buried in a protein.
d) The fluorescence of Trp changes depending upon the polarity of its environment.

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14. Which ONE of the following statements concerning the amino acid Histidine is
INCORRECT?
a) Its side-chain has a pKa of about 6.5, making it both a good hydrogen bond donor and
acceptor at neutral pH.
b) Poly-His sequences bind to Ni-affinity columns and are commonly used tag to purify
recombinant proteins expressed in bacteria.
c) The mutation of Histidine to Alanine in the active site of the enzyme ribonuclease A
results in a loss of enzyme activity.
d) It is classified as an aromatic amino acid because it contains an imidazole ring.

15. You are working on hemoglobin (Hb) S and are interested in studying the effect of
other amino acid substitutions at position 6 other that Glu6Val. You find that Glu6Ala
Hb and Glu6Lys do not aggregate, but that Glu6Ile Hb does.
Which ONE of the following interpretations is INCORRECT?
a) Ala is less hydrophobic than Val, so an Ala mutant of Hb is less likely to aggregate.
b) Lys is a strong helix former and is positively charged like Glu.

c) Ile is hydrophobic and -branched like Val so the Ile mutant would have a similar
effect on Hb as the Val mutant.

d) Ile is a strong -strand former so the Glu6Ile mutant would have a similar effect on the
properties of Hb as the Val mutant.

16. You are studying the stability of Ribonuclease A containing a single, buried
tryptophan residue using the protein denaturant urea. You perform fluorescence, circular
dichroism (CD) and infrared (IR) spectroscopy to study the effect of urea on the structure
of the protein.
Which ONE of the following predictions is INCORRECT?
a) The CD and IR spectroscopy can be used to measure changes in secondary structure of
the protein upon denaturation.
b) The fluorescence of the Trp residue in the protein will decrease and red-shift upon
denaturation.
c) Complete denaturation of Ribonuclease A in urea cause a dramatic change in the CD
spectrum of the protein.
d) Removal of urea in the presence of small amounts of reducing agent will not result in
any change in the fluorescence of the Trp residue.

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17. Which ONE of the following statements concerning fibrous proteins is
INCORRECT?
a) Collagen contains a low content of proline and hydroxy-proline, and forms a typical -
helix with 3.6 residues per turn.
b) Silk contains a high content of glycine within a 6-residue repeating unit that forms a -
pleated sheet, making its fibers highly flexible.
c) Wool (keratin) has a low glycine content and can be stretched via a helix to -sheet
transition, with disulfide crosslinks providing the main restoring force.
d) Actin exists as a monomer (G-actin) and in long filaments (F-actin) of multiple
subunits that make up the cytoskeleton of cells, giving them their shape.

18. Which ONE the following statements concerning the peptide bond is INCORRECT?
a) It is formed during protein biosynthesis in the ribosome in a condensation reaction that
releases water.
b) It has some double-bond character and therefore is not free to rotate.
c) It has a rigid planar structure and the amide hydrogen and carbonyl oxygen cannot
participate in hydrogen-bonding with other atoms.
d) It has a trans conformation resulting in the alpha carbons being 180° (omega angle)
from each other.

19. Which ONE of the following statements concerning the methods used to separate
proteins is INCORRECT?
a) SDS gel electrophoresis separates proteins based on the molecular mass of their subunits.
b) Gel filtration chromatography separates proteins based on their molecular size and
shape.
c) Ion exchange chromatography separates proteins based on their net surface charge.
d) HPLC reverse-phase chromatography separates proteins based on their affinity for a
specific ligand like a phenyl or octyl group bound to resin.

20. Which ONE of the following spectroscopic methods is INCORRECT?

a) CD spectroscopy can be used to determine the-helix and -sheet content in proteins.
b) Fluorescence spectroscopy can be used to determine the-helix and -sheet content in
proteins.
c) IR spectroscopy can be used to determine the-helix and -sheet content in proteins.
d) NMR spectroscopy can be used to determine the-helix and -sheet content in proteins.

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Part 3: You are a summer student work in a biochemistry lab. Answer
each of the following relevant questions with your ONE BEST answer.
21. You are synthesizing a small 12-residue peptide and mistakenly use Leu instead of
Lys in one of the steps in your reaction.
What effect would this have on the peptide?
a) The peptide would be less helical.
b) It could introduce a reverse turn into the peptide.
c) The peptide would be less water-soluble.
d) Nothing, it wouldn’t make any difference.

22. You are expressing a GST fusion with your peptide of interest in E. coli.
What essential step you would use to purify your protein of interest?
a) Trypsin digestion
b) GST affinity chromatography
c) Ion exchange chromatography
d) Western blot

23. You are designing a peptide with a helix-reverse turn-helix conformation.

Which sequence would you choose for your design?
a) GluAlaAlaAlaAsnProGlyGlyAlaAlaAlaLys
b) ValIleValIleAsnProGlyGlyValIleValIle
c) CysAlaAlaAlaAsnProGlyGlyAlaAlaAlaCys
d) AlaGlyAlaGlyAsnProGlyGlyAlaGlyAlaGly

24. Urea is a strong protein denaturant because:

a) it can break hydrogen bonds in proteins.
b) it precipitates proteins from solution causing them to aggregate.
c) it is a very hydrophobic molecule.
d) it can reduce disulfide bonds.

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25. What effect would adding small amounts of a non-polar solvent to a protein in
aqueous solution have?
a) No effect.
b) The protein would be more soluble.

c) The protein would undergo -helix to -strand transition.

d) The protein would be less stable.

26. You have isolated a 11-residue peptide with the amino acid composition Ala7, Trp1,
Arg2, Met1.
Cyanogen bromide treatment produces two fragments, one with the sequence
AlaAlaAlaMet.
Chymotrypsin treatment also produces two fragments, one peptide with the sequence
AlaAla.
Trypsin treatment produces three fragments, the amino acid Arg, and one peptide with the
sequence TrpAlaAla.
Which ONE of the following is the CORRECT sequence of this peptide?
a) AlaAlaAlaMetAlaAlaArgArgTrpAlaAla
b) AlaAlaArgArgAlaAlaAlaMetTrpAlaAla
c) AlaAlaArgArgTrpAlaAlaAlaAlaAlaMet
d) AlaAlaAlaMetTrpAlaAlaAlaAlaArgArg

27. You are asked to design a more temperature-stable version of a soluble enzyme using
site-directed mutagenesis.
Which ONE of the following design principles would you follow?
a) Larger hydrophobic residues should be placed in the core of the protein.
b) Mutating the active site to produce an inactive enzyme will stabilize the enzyme.
c) Charged residues should be introduced on the protein surface.
d) A single cysteine residue should be added to allow the protein to become oxidized.

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28. You are studying -helix/-strand transitions in a model peptide with the sequence
Val-His-Leu-Thr-Pro-Glu that has equal propensity to form an -helix or a -strand. An
equal mixture of the two conformations in aqueous solution confirms this propensity.
Which of ONE of the following peptides containing a single amino acid change would be
MORE likely to form a helix?
a) Val-His-Leu-Thr-Pro-Val
b) Val-His-Ile-Thr-Pro-Glu
c) Ile-His-Leu-Thr-Pro-Glu
d) Val-His-Leu-Thr-Ala-Glu

29. You discover a new mutation in the enzyme ribonuclease, which changes a Valine
that is completely buried in the protein to a Glutamate causing the protein to misfold and
become inactive. You decide to replace Valine with all other 19 amino acids. Of the
following four mutants which ONE would you predict to have the BEST activity:
a) Aspartate
b) Alanine
c) Arginine
d) Proline

30. The human hormone glucagon has the amino acid sequence:
H1SQGTFTSDY10SKYLDSRRAQ20DFVQWLMNT29
The underlined sequence from residue 18 to 27 can assume a helical conformation when
released from the pancreas into the blood and when bound by the glucagon receptor. It was
noted that the side-chain of D21 forms an ionic bond with the side-chain of R18 one helical
turn away, while R17 forms an essential ionic bond with D208 in the receptor.
You are working for a drug company and are asked to design a super-glucagon that is more
water-soluble with a more stable helical region.
Which ONE of the following statements would you provide to your research supervisor as a
rationale for the peptide you would design?
a) D21 should be mutated to E21, which would also maintain the ionic bond with R18.
b) D21 should be mutated to A21, which would also break the ionic bond with R18.
c) W25 should be mutated A25, which would allow tighter binding to the receptor.
d) Q20 and Q24 should be changed to C20 and C24, so that a disulfide bond can form.

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Part 4: Answer the following relevant questions based on the structure
of Insulin.

The above figure shows the ribbon structure of the storage form of the hormone insulin
crystallized in the presence of the ion Zn2+, shown as a central sphere. Based on your
examination of the structure, you correctly conclude that insulin is a highly -helical
protein that crystallized as a hexamer with six histidine side chains holding the central
Zn2+ ion in place.
Each subunit is composed of an A and B chain linked together by disulfide bonds as
shown below:

You are asked to design experiments to test the following features:

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31. In order to confirm the oligomeric structure of the protein you should perform:
a) circular dichroism measurements
b) gel filtration analysis
c) histidine-mutagenesis
d) Anfinsen’s refolding experiments

32. In order to confirm the secondary structure of the protein you should perform:
a) circular dichroism measurements
b) Chou-Fasman analysis of the sequence
c) Mass spectrometry
d) Fluorescence spectroscopy

33. In order to confirm the role specific histidines play in binding Zn2+ you should perform:
a) a pH titration of Zn2+ binding
b) Fluorescence spectroscopy
c) site-directed mutagenesis of His
d) NMR

34. Treating insulin with trypsin results in loss of activity. Which ONE of the following
BEST explains this phenomena?
a) It dissociates the hexamer.
b) The B-chain is cleaved at Arg and Lys residues.
c) The A-chain is cleaved at aromatic residues.
d) It cleaves disulfide bonds.

35. You express the two insulin chains together in E. coli but obtain an inactive form.
Which ONE of the following statements BEST explains your results?
a) E. coli doesn't normally make insulin.
b) E. coli cannot secret insulin.
c) E. coli cannot make the proper disulfide bonds.
d) E. coli cleaves insulin into a smaller peptide.

-END-

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