Biochem 4420A Assignment #1 - Total 50 marks (15% of final mark)

The assignment is due at 4:00pm on Thursday, October 9. Assignment must be

submitted via OWL. Penalties of 10% per day apply to all late submissions.
Following is a worked example:

The students original mark is 40.
Mark after penalty
up to 24 hours late 36
up to 48 hours late 32
up to 72 hours late 28
up to 96 hours late 24
up to 120 hours late 20
up to 144 hours late 16
up to 168 hours late 12
up to 192 hours late 8
up to 216 hours late 4
late for more than 216 hours 0

Question 1 (16 marks)

Protein C is a small monomeric protein. DSC experiments have been performed on
Protein C from a mesophile, Pseudomonas aeruginosa (P. aeruginosa), and a
thermophile, Hydrogenobacter thermophilus (H. thermophilus). The table below shows
the data obtained from these experiments. !C
p
= C
p
(unfolded) C
p
(folded).

T
m

(
o
C)
!H
unfold
(T
m
)
(kcal mol
-1
)
!C
p

(cal mol
-1
K
-1
)
Protein C from
P. aeruginosa
62.6 60.8 718
Protein C from
H. thermophilus
94.0 69.5 542

Plot !G
unfold
(T) versus T (T = 150 to 380 K) for P. aeruginosa and H. thermophilus
Protein C, assuming that the !C
p
values are constant over the temperature range (6
marks). Determine the temperatures of a) cold denaturation (2 marks) and b) maximum
stability (2 marks) for P. aeruginosa and H. thermophilus Protein C. Determine the
!!G
unfold
(!!G
unfold
= DG
unfold
(P. aeruginosa) - DG
unfold
(H. thermophilus)) at 37
o
C (2
marks).

An alanine residue located within the core of H. thermophilus protein C was mutated to a
valine by site-directed mutagenesis. The resulting mutational variant has the same T
m
and
!H
unfold
(T
m
) as the wild type. However, the !Cp of the mutant is increased to 600 cal
mol
-1
K
-1
. Is the mutant more stable than the wild type at 37
o
C (2 marks)? What is the
cold denaturation temperature of this mutant (2 marks)?




Question 2 (10 marks)

V157F, an oncogenic mutation of p53, has been shown to significantly decrease the
stability of the core domain of p53. Crystal structure of this mutant reveals that even
though the mutation does not change the overall fold of p53, the side-chains within the
hydrophobic core of the protein are re-arranged. On the other hand, the N239Y mutation
can enhance the stability of p53. Urea-induced denaturations of the V157F and N239Y
mutants of p53 were monitored by fluorescence at 10
o
C. The table below shows the
fraction of protein unfolded at each urea concentration of these two mutants.

Urea (M) V157F N239Y
1.2 0.030
1.4 0.093
1.6 0.259
1.7 0.376
1.8 0.530
1.9 0.666
2.0 0.780
2.1 0.873
2.2 0.921
2.3 0.960
2.4 0.975 0.029
2.6 0.088
2.8 0.998 0.260
2.9 0.391
3.0 0.550
3.1 0.682
3.2 0.812
3.3 0.885
3.4 0.930
3.6 0.980
3.8 0.995

I. Determine the !G
unfold
(H
2
O) at 10
o
C and the constant of proportionality (m
D-F
) of the
V157F mutant using the data provided above (4 marks). Determine the urea
concentration at which half of the protein is folded (3 marks). Show the calculation
for your answer.

II. The !G
unfold
(H
2
O) of the core domain of wild-type p53 was determined to be 9.5
kcal/mol at 10
o
C. What is the stability difference between the wild-type and the
N239Y mutant of p53 (in kcal/mol) at 10
o
C? (3 marks). Show the calculation for
your answer.




Question 3 (24 marks)

Nrf2 is a transcription factor that plays a central role in coordinating the cellular
responses to oxidative stress. Under homeostatic conditions, the activity of Nrf2 is
repressed by Keap1. Upon binding to the Kelch domain of Keap1, Nrf2 is ubiquitinated
and is subsequently targeted for degradation. The structure of the Kelch domain of mouse
Keap1 bound to a segment of Nrf2 has been solved by X-ray crystallography (PDB code:
1X2R). The !G and !H of binding at 25
o
C were found to be -9.2 kcal/mol and -14.8
kcal/mol, respectively.

I. Use the webserver PISA (!""#$%%&&&'()*'+,'-.%#/)(%#*0+%) to analysis the
binding interfaces of this complex. What is the total buried surface area upon
complex formation (2 marks)?
II. Briefly discuss the major factors that contribute to the enthalpy and entropy
changes upon the complex formation. (8 marks)
III. G430C is a somatic mutation of Keap1 identified in some lung cancer
patients. Use the SDM prediction server
(http://mordred.bioc.cam.ac.uk/~sdm/sdm.php) to predict the !!G
fold

(!!G
fold
=DG
fold
(wild-type) DG
fold
(mutant)) of this mutation (2 marks).
Use the structure of the Kelch domain of mouse Keap1 in the absence of
binding partner (PDB code: 1X2J) for the prediction.
IV. Based on the 1X2J structure, speculate on the possible origins of the
destabilizing effects of the G430C mutation (8 marks).
V. R415G is another somatic mutation of Keap1 that has been shown to
significantly impair the Nrf2-repression function of Keap1. However, SDM
predicts that this particular mutation does not affect the stability of Keap1.
Based on the 1X2R structure, speculate on the effects of R415G mutation on
Keap1-Nrf2 binding (4 marks).