7.

02 PBC exam study guide Fall 2001 1

Protein Biochemistry Study Guide--Fall 2001

In the past, the exam has consisted of ~40% “lecture” material and 60% “lab”
material. Thus, you should spend time with both your lecture notes, recitation notes,
and lab manual to prepare for the exam. (Don’t worry, we will NOT expect you to
derive the Michaelis-Menten equation. However, you should know what Km, Vmax,
and Kcat are!!). This list is not meant to be exhaustive, but to get you thinking about
things to study! Attached to this list is a set of study questions taken from old exams, as
well as the Spring 2001 exam. Use these to prepare for your exam on October 30, 2001.

“Lecture Topics”

• Properties of amino acids. We expect you to know the structure of the amino acids
and their properties (charged, hydrophobic, polar, H bond donor or acceptor, etc.)
• pKa of an amino acid’s side chain, isoelectric point (IEP) of a protein—what do these
numbers mean? What are the properties of the side chains of the above amino acids
above and below their pKas? What are the properties of a protein above and below
its IEP?
• The famous, planar peptide bond—How is it formed? (What is the chemical
reaction?) What groups (NH3, COOH, etc.) are in the plane of the peptide bond?
• Primary, secondary, tertiary, and quaternary structure. What is meant by each of
these terms? Examples of each from lecture or lab.
• Alpha helices—what they are, where does hydrogen bonding occur, are they right or
left handed, how does the presence of proline affect a helix, etc.?
• ß-sheets—what are they? What is the difference between a parallel and antiparallel
ß-sheet? Where does hydrogen bonding occur in these molecules? What’s a ß-barrel,
ß-saddle, TIM barrel?
• Examples of famous molecules that have alpha helices, ß-sheets, ß-barrels, etc.
• Collagen—what it is, what it looks like, how structure affects function
• Antibodies—structure (heavy chain, light chain, constant domains, variable
domains), different secondary structural elements involved (helices, ß-barrels), what
parts are more variable and conserved, and how this impacts function, CDR
(complementarity determining regions); Generation of Diversity (“the GOD
problem”).
• GFP—structural elements, what is it used for, what features of the active site make it
a good fluorophore,
• ß-gal—what is its enzymatic activity, what is the quaternary structure like,
importance of Mg+2, domain structure, how are active sites formed structurally,
how many active sites, what are the important residues in the active site, and how
do they impact enzymatic activity
7.02 PBC exam study guide Fall 2001 2

“Lab Topics”: Basically, if it’s in the manual, it’s fair game!!

Purification and Analysis methods/Techniques:

• Lysing cells—methods? What reagents affect?
• Ammonium sulfate ppt—how it works?
• Gel Filtration—how it separates, properties of matrix, what determines
elution order, method of elution?
• Ion-Exchange—how it separates, properties of matrix, what determines
elution order, method of elution?
• Affinity Chromatography—how it separates, properties of matrix, what
determines elution order, method of elution?
• SDS-PAGE, Coomassie staining
• Western blots-primary, secondary antibodies, alkaline phosphatase reaction,
nitrocellulose, whatman paper
• Bradford (Biorad) Assay
• ß-gal assays (substrate, reaction catalyzed)

Reagents used in our experiments/talked about in recitation

• lysozyme
• hypotonic buffer
• DNase, MgCl2
• Ammonium sulfate
• DEAE, Tris, NaCl, MgCl2, DTT
• APTG, ONPG, Xgal (ok, in genetics, but still relevant!), NaCO3
• GST, MBP, (HIS)6
• SDS, ß-mercaptoethanol (ß-ME), glycerol, bromophenol blue
• acrylamide
• Coomassie
• Blotto
• Washing during westerns
• Primary antibodies, secondary antibodies, alkaline phosphatase
• BCIP, NBT

Calculations—the ones you did in lab!

• Rate of reaction (velocity)
• Total activity
• Specific Activity—how to calculate it, what it means, how it changes during a
purification
• Fold purification—what it is, how to calculate it
• Percent Yield—what it is, how to calculate it, how it changes during a
purification
• Determining [Etot]
• Kcat, Km,Vmax from initial velocity, substrate concentrations, and total
protein

In general, besides knowing the “facts” about these lab techniques, think about how
they can be used and adapted to solve a problem of interest!
7.02 PBC exam study guide Fall 2001 3

Study questions for 7.02 PBC Exam

Fall 2001

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 ______ a) has a charged residue at pH=7
threonine ______ b) non-polar/hydrophobic
proline ______ c) imino acid
phenylalanine ______ d) has a ring
cysteine ______ e) absorbs light at wavelength 280 nm
glutamic acid ______ f) contains sulfur
lysine ______ g) polar/hydrophilic
h) has the simplest side chain

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.

b) When loading a polyacrylamide gel, the sample diffused out of the well 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?

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

7.02 PBC exam study guide Fall 2001 4

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

b) circle all the regions of hydrogen bonding in your drawing

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: ___________, ____________, _________.

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

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

7.02 PBC exam study guide Fall 2001 5

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

b) APTG

c) nitrocellulose membrane

d) sodium carbonate (Na2CO3)

e) DTT
7.02 PBC exam study guide Fall 2001 6

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)

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

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
)

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).

1
7.02 PBC exam study guide Fall 2001 7

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:

Quaternary structure:

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

collagen: __________________
immunoglobulin (like IgG): ___________________
Green Fluorescent Protein (GFP): ______________

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)
7.02 PBC exam study guide Fall 2001 8

(Question 4 continued)

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

AS-P 4 10 40 120000

DEAE- 2 17.5 35 105000

Load
DEAE- 2 11 22 68200
Total
AF after 1 3 3 60000
PD-10

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).

b) (3 points) Which step of the protocol gave the best purification? How do you know?

c) (4 points) If Dr. Tics asked you to improve this protocol, which step might you
choose to eliminate? Why?
7.02 PBC exam study guide Fall 2001 9

Question #5 (25 points total)

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.

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?

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.
7.02 PBC exam study guide Fall 2001 10

(Question 5 continued)

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
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
7.02 PBC exam study guide Fall 2001 11

(Question 5 continued)

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?

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.
7.02 PBC exam study guide Fall 2001 12

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:

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!

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