Question

You are studying the amide hydrogen exchange (HX) of an alanine residue in an α- helix in a protein. You measure the rate of exchange by placing the protein in D2O (pH 6, 20°C) and observe the amide proton signal intensity decrease as a function of time. Why does the peak intensity diminish? HX follows the relationship It = Ioe-kt where It is the peak intensity at time t, Io is the initial intensity, and k is the exchange rate constant. Using a suitable graphic or least squares approach, determine the HX rate constant for the alanine from the following experimental results:

time (s) It

100 100

300 90

500 74

750 60

1000 47

1500 37

2000 22

Under these conditions, the HX rate constant of an alanine in an unstructured polypeptide chain is 10 s-1 . By how much is the exchange of the alanine amide retarded due to its location in the folded protein (e.g., what is its protection factor)? Provide a possible reason for this result.

Answer 1

Why does the peak intensity diminish?

It diminish because the interchange is taking place over the time and the technique is able to observe only to N-H bond and not the N-D bond.

The way to obtain the costant k is taking the ln both sides and linearize.

It = Io

taking ln both sides

ln(It) = ln(Io) -kt

plotting ln(It) vs t

where the slope is straigh the k = 0.0008.   know we take the ratio :

10 s^-1 / 0.0008 = 12500 fold retarded due its location in the folded protein.

One reason for this retardation is the formation of hydrogen bonds between the proton and another residues in the protein that difficult the interchange with the water.