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Study questions for 7.02 PBC Exam--Fall 2001

ANSWER KEY

1. Below are seven amino acids. Indicate all characteristics that apply to each amino
acid by writing letter(s) in the blanks provided.

Amino acid Characteristics

glycine __h, b___ a) has a charged residue at pH=7
threonine __g___ b) non-polar/hydrophobic
proline __c, d, (b) c) imino acid
phenylalanine __b, d, e_ d) has a ring
cysteine __f (b, g)_ e) absorbs light at wavelength 280 nm
glutamic acid __a, (g)__ f) contains sulfur
lysine __a, (g)__ g) polar/hydrophilic
h) has the simplest side chain

Answers in parentheses are acceptable but not necessary.

2. Little Johnny Biochemist was purifying some β-galactosidase using the 7.02 manual
the other day and he ran into the following problems. Explain why LJB’s experiments
didn’t work and recommend how he can fix each of his problems.

a) There’s β-gal in the flow-through of the DEAE column.

There are a number of correct answers here. Some include: too much protein in
the load, wrong pH buffer, too much salt in the column buffer or protein sample
(didn’t desalt after AS precipitation.

b) When loading a polyacrylamide gel, the sample diffused out of the well into the
running buffer.
Glycerol is important for increasing the viscosity of the sample buffer. This
prevents the samples from diffusing out into the running buffer.

c) An ammonium sulfate cut was made on a newly discovered protein called

Namedoesn’tmatter-ase. After LJB resuspended the pellet, he assayed it and found no
activity. A post-doc in the lab redid the ammonium sulfate step exactly like LJB and
used his reagents. The post-doc found active protein. What happened?

This is a tricky question. The point was that the protein can remain in the
supernatant and not just in the pellet. The enzyme, and thus the activity, was in the
supernatant. LJB didn’t test the sup, so he didn’t find any activity!

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3. Draw the peptide asp-val-glu-gly and circle the peptide bonds.

(see attached figures page)

4. Mark whether each of the following statements is true (T) or false (F).
_T__ a) Km equals [S] when the velocity of an enzyme-catalyzed reaction is 1/2 Vmax.
_F__ b) In gel filtration, smaller molecules elute first.
_F__ c) The repeating unit of collagen is ala-pro-hyp
_T__ d) The heavy chain of antibodies contains 1 variable region and 3 constant
regions
_T__ e) Sickle-cell anemia is caused by a single E6V mutation in the β chain of
hemoglobin.

5a) Draw an antiparallel β pleated sheet composed of 2 β strands, each with 4 amino
acids (use R1, R2, R3…., R8 for the side chains of the 8 amino acids.)

(see attached figures page)

b) circle all the regions of hydrogen bonding in your drawing. (see figures page)

c) one side of the sheet is found to be strongly hydrophobic. Give the full names of
three amino acids that can fulfill this property: _alanine__, __valine__, __leucine__

d) on your drawing, circle the R groups that have to be hydrophobic so that one side of
the pleated sheet is hydrophobic. (see figures page)

6. Briefly explain the use of SDS and β−mercaptoethanol in protein denaturation.

SDS in an amphipathic molecule consisting of a long carbon chain and a polar,

charged sulfate group. It denatures proteins by making hydrophobic interactions
with the amino acids in the proteins. It also coats the denatured proteins with a
negative charge. β-mercaptoethanol is a strong reducing agent which breaks
disulfide bonds (both inter- and intramolecular).

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Spring 2001 PBC exam

Question #1 (20 points total)

Describe the purpose of the following reagents in the PBC module. (A complete answer
will BRIEFLY describe when it was used in the module and what it is/how it works
/what it does) (4 points each).

a) lysozyme
• an enzyme (NOT a detergent or chemical)
• used on PBC Day 1 during the lysis of bacterial cells
• breaks down cell wall linkages (-0.5 for cell membrane)

b) APTG
• used on the affinity column during ß-gal purification
• substrate analog for ß-gal; ß-gal binds APTG specifically
• non-cleavable

c) nitrocellulose membrane
• used during Western blotting
• binds proteins nonspecifically
• proteins transferred by electrical current from the gel

d) sodium carbonate (Na2CO3)

• STOP solution for ß-gal assays
• Strong base; raises pH, unfolds/denatures ß-gal to stop reaction
• Also aids ONP color development

e) DTT
• (weak) reducing agent (NOT DETERGENT)
• used in buffers/solutions (column buffer)
• breaks non-native disulfides, promotes proper folding, simulates the
intracellular environment (slightly reducing)

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Question #2 (19 points total)

a) (8 points) Draw the structure of the following tetrapeptide at pH 7.0:

(Nterm) cysteine—tyrosine—lysine—glutamic acid (C term)

(see attached figures page)

Side chains (6 points)—1.5 points/AA; -0.5 for missing/extra carbon or otherwise

incorrect R group. Peptide bonds (2 points)—peptide bonds needed to be planar (i.e.
side chains alternating)

b) (1 point) Which of the four amino acids in this tetrapeptide can form
disulfide bonds? cysteine

c) (6 points) Alpha helices are stabilized by hydrogen bonding between “hydrogen

donors” and “hydrogen acceptors.” On your drawing in part a), label the atoms that
can serve as hydrogen donors with an arrow labeled “D” (D
) and those that can serve
as hydrogen acceptors with an arrow labeled “A” (A
) (see figures page)

3 points for side chain H bonders

3 points for backbone H bonders
-0.5 for cysteine

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d) (4 points). Below is a diagram of a cross-section of an alpha helix with the side chain
of amino acid 1 labeled “#1.” Based on what you know about the structure of alpha
helices, label the positions of the side chains of amino acids #2-7. (Assume that you are
looking down the axis of the helix, and that the N-terminal amino acid is closest to you).
Clockwise: 1 point
Correct order: 3 points
0.5 points/AA if “luckily” in right place

4 5

7 2

3 6

Question #3 (14 points total)

a) (8 points) Briefly define each of the following levels of protein structure, and give an
example of each from lecture or lab:

Secondary structure:
Definition (3 points) – local conformation of backbone without regard to side
chains
Example (1 point) – alpha helix (beta sheet or turn).

• Quaternary Structure

Definition (3 points) – spatial arrangement of subunits (subunit = 1 polypeptide).

This occurs by noncovalent interactions and sometimes disulfide bonds.

Example (1 point) – 4 subunits of hemoglobin, etc.

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b) (6 points) Name the major tertiary structural element in each of the following
molecules:

collagen: superhelix, coiled coil (3 alpha helices wrapped around each other)
immunoglobulin (like IgG): flattened ß-barrel or ß-barrel
Green Fluorescent Protein (GFP): ß-barrel

Question #4 (12 points total)

After 7.02 ends, you decide to do a UROP in the lab of Dr. Jean Tics. Dr. Tics wants to
see how much you learned about protein purification in 7.02, so she asks you to purify a
well characterized protein (PHD Isomerase) using the lab’s standard protocol.
Starting from crude lysate (CL), you performed the following steps (and created
these samples to be assayed):

1. Precipitation by ammonium sulfate (yielding AS-P)

2. Desalting on PD-10 column (yielding DEAE-Load)
3. DEAE column (eluted fractions pooled as DEAE total)
4. Affinity column followed by PD-10 desalting (yielding AF after PD10)

You then assayed each sample for PHD Isomerase activity, and obtained the
following results:

sample Volume Protein Total Total Specific Fold

(ml) conc. protein Activity Activity Purification
(mg/ml) (mg) (U)
CL 25 4 100 200000 2000 1X

AS-P 4 10 40 120000 3000 1.5X

DEAE- 2 17.5 35 105000 3000 1.5X

Load
DEAE- 2 11 22 68200 3100 1.55X
Total
AF after 1 3 3 60000 20000 10X
PD-10

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a) (5 points) Fill in the specific activity and fold purification for each step of the
purification on the chart above (include units where appropriate).

Specific Activity: Units= U/mg (-0.5 points if no units); 0.5 points each SA
Fold Purification (no units)--0.5 points each

b) (3 points) Which step of the protocol gave the best purification? How do you know?
The affinity column (or Step #4) gave the best purification (1 point). You can tell this
because this step had the best fold purification (2 points).

c) (4 points) If Dr. Tics asked you to improve this protocol, which step might you
choose to eliminate? Why?

You could improve the protocol by eliminating the DEAE column (Step #3) (1 point).
This column gives you no real increase in SA or fold purification, and results in a
significant loss of protein (3 points).

Convinced that you are an expert in protein purification, Dr. Tics presents you with
your next challenge: purifying and characterizing wild type and mutant forms of the
enzyme “NoPBCase.” After preparing your crude lysate (CL), you decide to precipitate
“NoPBCase” with ammonium sulfate.

a) (4 points) Briefly describe how ammonium sulfate precipitates proteins.

In solution, proteins are surrounded by solvation cages (hydrogen bonding and ionic
interactions between the water and the surface charges of the protein). Adding high
concentrations of salt interferes with/breaks these cages (water interacts with AS
instead of protein) (2 points). This allows the protein molecules to interact with each
other, form aggregates, and precipitate out of solution (2 points).
(1 point) For saying “salting out.” Partial credit was also given for less complete
descriptions of the process.

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The postdoc you are working with has done some preliminary work with
NoPBCase, and she has determined that 100% of the NoPBCase in the crude lysate will
precipitate at 40% ammonium sulfate. Unfortunately, you make an error in your
calculation of how much AS to add, and end up bringing your crude lysate to 60%
ammonium sulfate!

b) (3 points) Do you expect that this mistake will affect the total activity (T.A.) of
NoPBCase that you obtained in your AS-P fraction (increase, decrease, no change)?
Why or why not? (1 point for answer, 2 points for reasoning)
My answer: If 100% of the NoPBCase is precipitated at 40% AS, then we’d not expect
more enzyme to precipitate at 60% AS. Since TA reflects the activity of the enzyme
of interest, then we’d expect to see no change in TA
Credit was also given if one thought TA would decrease because of denaturation
(assuming that high salt killed your enzymatic activity).

c) (3 points) How will this mistake likely affect the specific activity (S.A.) of your AS-P
fraction (increase, decrease, no change)? Explain your answer. (1 point for answer, 2
points for reasoning)

Specific Activity= Total Activity/Total Protein. Total activity should not change
because of 60% AS addition. However, by adding 60% AS you will precipitate many
more proteins out of solution. Therefore, total protein concentration will increase,
and specific activity (“purity”) will decrease.
If you said that TA would decrease because of denaturation of your enzyme, you
would also have to mention protein concentration increases to get full credit here.

You decide that ion-exchange chromatography is the next logical next step in your
purification. Since you know very little about the properties of NoPBCase , you decide
to try both an anion exchanger (DEAE-Sephacel) and a cation exchanger
(carboxymethyl (CM)-cellulose. You apply half of your desalted AS-P to each column
and collect the flowthrough (FT). You then add increasing amounts of salt to elute any
proteins stuck to the columns, and collect five fractions (1-5). Finally, you perform a

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quantitative assay of each fraction for activity (The reaction catalyzed by NoPBCase
produces a colored product whose accumulation can be measured at A420).

d) (7 points) On the following plots of A420 vs. fraction number, indicate where you’d
expect to see the peak of NoPBCase activity if NoPBCase is positively charged in the
buffer conditions used for each column. Briefly explain your reasoning.

DEAE-Sephacel CM-Cellulose
A420 activity

A420activity

FT FT 1 2 3 4 5 FT FT 1 2 3 4 5

begin adding begin adding

increasing salt increasing salt
concentrations concentrations

(1.5 points for each correct graph)

(4 points for reasoning)

DEAE Sephacel and other anion exchangers will bind to negatively charged
proteins. Thus, the positively charged NoPBCase will be found in the FT off this
column. CM cellulose is a cation exchanger, and thus binds positively charged
proteins like NoPBCase. It will likely be eluted in the middle of the salt gradient
(where depends on its net positive charge). Elution occurs when positively charged
ions in the buffer compete with the immobilized proteins for the negatively charged
matrix.

If you mixed up cation and anion exchangers, but your reasoning was right based on
your misunderstanding, you received 2 points.

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Finally, you decide to characterize the “size” and structure of NoPBCase. You find
that NoPBCase behaves like a 120 kDa protein on a gel filtration column (elutes at the
same time as a 120 kDa protein standard). You and your postdoc next decide to do
Western blots on your purified protein sample. You both run SDS-PAGE gels, and
perform Western blots together using anti-NoPBCase antibodies. You observe the
following:
your
your postdoc's
gel gel

120 kDa 120 kDa

100 kDa 100 kDa
80 kDa 80 kDa

40 kDa 40 kDa

e) (2 points) You and your postdoc analyzed the same protein sample on your gels.
Why are your results different?

When preparing your samples for SDS-PAGE, you forgot to add reducing agent to
your sample (ß-ME). Your postdoc added ß-ME, which reduces disulfide bonds
between multimeric proteins as well as intramolecular disulfides. Thus, her sample
was fully reduced—and separated into monomers—while yours was not (and
remained in the trimer form). (1 point was awarded for recognition of monomers vs.
trimers, but wrong reasoning)

f) (6 points) Propose a model for the structure of NoPBCase that takes into account all
of the gel filtration and SDS-PAGE data. Justify your answer.
Gel filtration measures the size of the native form of the protein (120 kDa). Addition
of reducing agent changes the apparent MW to 40 kDa, and results in 3X as much
protein on the Western blot. This is consistent with a model of a positively charged,
trimeric protein consisting of 40 kDa monomers held together by disulfide bonds.

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Question #6 (10 points total)

You finally have some purified NoPBCase and are ready to do some enzyme
kinetics. In your assays, you make use of ONPB, an analog of NoPBCase’s natural
substrate (NoPBCase catalyzes the following reaction: ONPB
ONP + Biochemose).
You perform an enzyme kinetics experiment (like in 7.02 lab) using different
concentrations of ONPB substrate (and measuring ONP production at A420). Here is the
data you collected from your first experiment with wild type NoPBCase:
mutant defective
in substrate
binding
0.25

0.2
1/ v ( ml/ nmol ONP/ min)

0.15

0.1

0.05

0
-60 -40 -20 0 20 40 60 80 100 120
1/ s (ml/ nmole)

a) (6 points) Determine Km and Vmax for wild type NoPBCase. Show your work, and
don’t forget units! (2 points each for correct calculation/ 1 point each for correct units)
On a Lineweaver-Burk plot, the X intercept is equal to –1/Km, while the Y intercept is
equal to 1/Vmax. Therefore:
-1/Km = -32 ml/nmole and Km = 0.033 nmole/ml
1/Vmax = 0.055 ml/nmole/min and Vmax = 16.7 nmole ONP/ml/min

b) (4 points) The postdoc that you are working with has purified a mutant version of
NoPBCase. She is not sure whether the mutation affects substrate binding, catalysis, or
both. On the Lineweaver-Burk plot in part a), draw the results you’d expect if the
mutation caused a defect in substrate binding. Km should increase (-1/Km becomes
more positive) and there should be no change in Vmax.

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