Question

1.

A novel protein is described that functions best at pH 11.5 (!), and contains a loop between helix 2 and helix 5 of the protein. The authors report that at pH 11.5 this loop is stabilized by formation of a salt bridge between two amino acid side chains.

a. Which of the following amino acid pairs would be the best candidates for this interaction and why: GlnCys, Tyr-Arg, or His-Asp.

b. Show the full amino acid structures and interactions that support your answer.

2.

Use the table below, which describes properties of several hypothetical enzymes, to answer the following questions. Prior to purification all four enzymes are present in a single sample

. a. What chromatography method would best separate One-ase from Two-ase?

b. What chromatography method could be used to separate Two-ase from Six-ase?

c. If the mixture of all six enzymes was separated by first using the method you chose in (a) followed by the method you chose in (b) would Five-ase be separated from all the other enzymes?

d. Show the bands present for each protein under non-reducing and reducing conditions on both a native PAGE and SDS PAGE gel (draw a separate lane for each protein on both gels).

e. In a mix of these six proteins, which will elute first from a native gel filtration column?

Protein	pI	Subunit composition & Molecular Weight
One-ase	6.0	One @ 20kD
Two-ase	9.5	One @20kD
Three-ase	9.5	One @40kD
Four-ase	7.5	Two @20kD
Five-ase	9.4	One @80kD
Six-ase	9.3	Four @20kD

Answer 1

answer 1.

answer 2:

a. chromatography method would best separate One-ase from Two-ase would be ion exchange chromatography.

b. chromatography method could be used to separate Two-ase from Six-ase- 2dimensional gel electrophoresis which is separated in 2 sequential step:first by their charge and then by their mass. 2DGE= isoelectric focusing+ SDS electrophoresis.

c. If the mixture of all six enzymes was separated by first using the method you chose in (a) followed by the method you chose in (b) would Five-ase be separated from all the other enzymes- yes , as we can see in the table the molecular weight of five-ase is higher, it can be separated by SDS page.

d.

e. In a mix of these six proteins, which will elute first from a native gel filtration column- five-ase

hope u got the points!