Biochemistry Practice Problems X with Answers

QUESTION 1
The idea that primary sequence determines tertiary structure first came from experiments in the 1950’s about:
A. the renaturation of RNaseA (ribonuclease A).
B. the denaturation of lysozyme.
C. the 3D structure of lysozyme.
D. the role of Hsp70 in protein folding.
E. the role of PDI (protein disulfide isomerase) in protein folding.

QUESTION 2
Questions #2 & 3 refer to the following information. Altered conformations of the prion protein (PrP) are implicated in the infectivity and/or pathogenesis of Scrapie-like diseases, such as “mad cow disease” and Creutzfeldt-Jacob Disease (CJD). Below are ribbon diagrams of two different conformations of PrP:
Which of the conformations illustrated above (a or b) is more likely PrPc (the normal cellular form of PrP) and which is more likely PrPSc (the pathogenic form of PrP)? Why?

QUESTION 3
The red arrows within the (b) conformation are best categorized as:
A. alpha helices
B. beta-turns
C. tertiary structure
D. parallel beta-strands
E. antiparallel beta-strands

QUESTION 4
Questions #4-7 refer to the figure shown below. Approach each question independently.
Figure Legend: Tagging schematic. The figure shows various steps involved in the identification of interacting proteins by using an epitope-tagging strategy. The cDNA of interest is first cloned into a vector that provides an epitope tag. This is followed by transfection of the tagged “bait” into the cell of interest. The cells are then lysed and the lysates purified by affinity purification using an antibody against the epitope. Proteins bound specifically to the bait protein are eluted by competitive elution using a peptide that encodes the epitope. The proteins are then resolved by gel electrophoresis followed by mass spectrometric identification.
STEP 1: Epitope-tagged protein (encoded by cDNA)
STEP 2: Transfect into cells
STEP 3: Lyse cells
STEP 4: Affinity-based purification using immobilized antibody against the epitope
STEP 5: Competitive elution with peptide encoding the epitope
STEP 6: Gel electrophoresis
STEP 7: Excise bands and analyze by mass spectrometry

QUESTION 4

According to the gel (Step 6), which protein has the highest molecular weight?
A. Protein A
B. Protein B
C. Protein C
D. Protein D
E. YFP

QUESTION 5
Are the bands indicated in Step 6 (SDS-PAGE) likely visualized by western blot or by a general protein stain (such as Coomassie or silver)? Explain your answer.

QUESTION 6
Step 7 states “Excise bands and analyze by mass spectrometry.”
(A) Following excision of the bands (simply cutting each band out of the gel), briefly describe each step, in order, that must be taken prior to analysis in the mass spectrometer.

(B) What’s the big experimental question that the researchers hope to answer following Step 7?

QUESTION 7
Following Step 5 (“competitive elution with peptide encoding the epitope”), it appears that the YFP/protein complex has dissociated — all proteins are drawn floating freely. Which of the following is the most likely explanation for dissociation of the protein complex?
A. Treatment of the eluate (Step 5) with beta-mercaptoethanol, in preparation for loading onto the gel (Step 6)
B. Treatment of the eluate (Step 5) with dithiothreitol, in preparation for loading onto the gel (Step 6)
C. Treatment of the eluate (Step 5) with sodium dodecyl sulfate, in preparation for loading onto the gel (Step 6)
D. Exposure of the complex to free peptide encoding the epitope (in other words, Step 5, the elution process itself)
E. Placing the eluate (Step 5) on ice, prior to loading onto the gel (Step 6)

QUESTION 8
A colleague has successfully purified the enzyme sphingomyelinase from bovine brain. She provides you with the following incomplete purification table:

Per your colleague’s request, you agree to calculate:
(A) the final specific activity,
(B) the final % yield, and
(C) the final fold-purification.

QUESTION 9
For the table shown in Question #8, the purification step labeled “40-60% (NH4)2SO4” is most likely a(n):
A. salting in/out step.
B. cation exchange chromatography.
C. anion exchange chromatography.
D. hydrophobic interaction chromatography.
E. size exclusion chromatography.

QUESTION 10
For the table shown in Question #8, the purification step labeled “CM-sepharose” is most likely a(n):
A. salting in/out step.
B. cation exchange chromatography.
C. anion exchange chromatography.
D. hydrophobic interaction chromatography.
E. size exclusion chromatography.

QUESTION 11
For the table shown in Question #8, the purification step labeled “Gel filtration” is most likely a(n):
A. salting in/out step.
B. cation exchange chromatography.
C. anion exchange chromatography.

D. hydrophobic interaction chromatography.
E. size exclusion chromatography.


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