BIS 102 Name ______________Key_______________________

Fall 2003 Last First

K. Hilt

Second Midterm Score (100): ___________

1. (24 pts.) What would the elution profiles look like for the first three cycles of Edman chemistry

for the protein sequenator of the following peptide: KAEFA ? Label your axes. Label each peak.

Label each cycle. Explain briefly why you drew the plot(s) the way you did.

Cycle #1 (+2) Cycle #2 (+2) Cycle #3 (+2)

Plot(s): bis-PTH-K (+2) PTH-A (+1) PTH-E (+1)

(+2) (+2) (+1) (+1)

Time (+2)

Reasoning: The column is hydrophobic (+2) and is being eluted with a gradient of polar to nonpolar

solvent (+2). Hence, hydrophobic compounds elute last (+2). Bis-PTH-K is doubly labeled and hence

gives a peak size that is twice that of PTH-A or PTH-E.

2. (14 pts.) A protein was completely digested with trypsin and an internal peptide (i.e. not

derived from the N- or C-terminals) was purified. Determine its sequence from the following

information:

a) amino acid analysis yields one mole of F, K, P, Y, G, R, and W per mole of peptide; b) treatment of

the intact peptide with Sanger’s reagent, followed by acid hydrolysis, gives DNP-Y; c) treatment of the

intact peptide with chymotrypsin yields free W, free Y, free R, and a tetrapeptide containing P, G, F,

and K; and d) treatment of the tetrapeptide in part "c" with carboxypeptidases A and B yields a large

amount of F and a smaller amount of G.

Note: trypsin will not cleave on the carboxyl side of R or K if the next amino acid (donating the

amino group) is P.

(+2) for each a.a. residue

Answer: ___Y-K-P-G-F-W-R or Y-W-K-P-G-F-R_____________

3. (14 pts.) Is it likely that the above sequence, in question #2, would participate in any type of 2o

protein structure? Identify each type of protein 2o structure that is possible in a protein and then discuss

whether the above sequence is likely to participate in that type. Give your reasoning.

Yes (+1). Possible 2o structures are -helix (+2), -pleated sheet (+2), or reverse turn (-bend)

(+2). P (+1) and G (+1) eliminate -helix. P (+1) eliminates -pleated sheet (+1). Reverse turn is

possible (+1) since P (+1) is followed by G (+1).

4. (14 pts.) Protein Z is denatured using 8 M urea and excess mercaptoethanol. The protein is then

allowed to renature. What do you do, experimentally, to allow the renaturation to occur? What is

happening, at the molecular level, in each step? What does the successful experiment prove?

First, dialyze (+2) the sample to remove the urea (+2). This allows the four weak bonds to

reform (+2). Secondly, bubble in O2 (+1). This allows disulfide bonds to reform (+1), if there are any.

Successful experiment proves: 1) all the information required for refolding is in the linear sequence of

a.a. residues (+3), 2) the folded 3o structure is the most thermodynamically stable form of the protein

(+3) .

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BIS 102 Name ______________Key_______________________ Fall 2003 Last First K. Hilt Second Midterm Score (100): ___________

(24 pts.) What would the elution profiles look like for the first three cycles of Edman chemistry for the protein sequenator of the following peptide: KAEFA? Label your axes. Label each peak. Label each cycle. Explain briefly why you drew the plot(s) the way you did. Cycle #1 (+2) Cycle #2 (+2) Cycle #3 (+2) Plot(s): bis-PTH-K (+2) PTH-A (+1) PTH-E (+1) A (+2) (+2) (+1) (+1) Time (+2) Reasoning: The column is hydrophobic (+2) and is being eluted with a gradient of polar to nonpolar solvent (+2). Hence, hydrophobic compounds elute last (+2). Bis-PTH-K is doubly labeled and hence gives a peak size that is twice that of PTH-A or PTH-E.
(14 pts.) A protein was completely digested with trypsin and an internal peptide (i.e. not derived from the N- or C-terminals) was purified. Determine its sequence from the following information: a) amino acid analysis yields one mole of F, K, P, Y, G, R, and W per mole of peptide; b) treatment of the intact peptide with Sanger’s reagent, followed by acid hydrolysis, gives DNP-Y; c) treatment of the intact peptide with chymotrypsin yields free W, free Y, free R, and a tetrapeptide containing P, G, F, and K; and d) treatment of the tetrapeptide in part "c" with carboxypeptidases A and B yields a large amount of F and a smaller amount of G. Note: trypsin will not cleave on the carboxyl side of R or K if the next amino acid (donating the amino group) is P. (+2) for each a.a. residue Answer: ___Y-K-P-G-F-W-R or Y-W-K-P-G-F-R_____________
(14 pts.) Is it likely that the above sequence, in question #2, would participate in any type of 2o protein structure? Identify each type of protein 2o^ structure that is possible in a protein and then discuss whether the above sequence is likely to participate in that type. Give your reasoning. Yes (+1). Possible 2o^ structures are -helix (+2), -pleated sheet (+2), or reverse turn (-bend) (+2). P (+1) and G (+1) eliminate -helix. P (+1) eliminates -pleated sheet (+1). Reverse turn is possible (+1) since P (+1) is followed by G (+1).
(14 pts.) Protein Z is denatured using 8 M urea and excess mercaptoethanol. The protein is then allowed to renature. What do you do, experimentally, to allow the renaturation to occur? What is happening, at the molecular level, in each step? What does the successful experiment prove? First, dialyze (+2) the sample to remove the urea (+2). This allows the four weak bonds to reform (+2). Secondly, bubble in O 2 (+1). This allows disulfide bonds to reform (+1), if there are any. Successful experiment proves: 1) all the information required for refolding is in the linear sequence of a.a. residues (+3), 2) the folded 3o^ structure is the most thermodynamically stable form of the protein (+3). 1 of 2

BIS 102 Name ______________Key___________________________

(34 pts.) A number of different test tubes of solutions were tested for their ability to bind oxygen. The solutions (labeled #1  #6) are described below. You are to draw the oxygen binding ) are described below. You are to draw the oxygen binding curves that you expect to see for each solution using the axes provided below. Label each axis. Label each curve. Briefly describe why each curve looks the way that it does. Solutions : #1 Mb in a buffer at pH 7. #2 Hb in a buffer at pH 7.6) are described below. You are to draw the oxygen binding. #3 Hb in a buffer at pH 7.6) are described below. You are to draw the oxygen binding in the presence of 50% saturating levels of carbon monoxide. #4 Hb in a buffer at pH 7.2. #5 Hb in a buffer at pH 7.2, but the C-terminal histidine on each of the -subunits has been replaced with an arginine #6) are described below. You are to draw the oxygen binding Hb in a buffer at pH 7.6) are described below. You are to draw the oxygen binding , but the C-terminal histidine on each of the -subunits has been replaced with a valine. 1 6) are described below. You are to draw the oxygen binding 2 5 4 (+3) for each curve % O 2 Bound 3 pO 2 Reasoning: Curve #1: Hyperbola (+1). Independent binding sites (+1), no communication Curve #2: Sigmoidal curve (+1). Communication between the four hemes (+1) Curve #3: CO binds 250 x stronger to the hemes, compared to O 2 (+1). Half of the heme sites are eliminated as O 2 binding sites. (+1) Curve #4: H+^ portion of the Bohr effect. Certain salt bridges are strengthened by the lower pH. Either answer (+2) Curve #5: ~ 40% of the H+^ Bohr effect is eliminated (+2). The pKa of R is far above the blood pH. Hence, it is always protonated. (+2) Curve #6) are described below. You are to draw the oxygen binding : Two, of the roughly 13 salt bridges in deoxy Hb have been eliminated (+2) with the change of H to V. (There are two -subunits). 2 of 2

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