[RASMB] dimer dissociation kinetics via sed. velocity

[Avi Chakrabartty]

Dear RASMB members,

We are working on the folding mechanism of a dimeric protein. Exposure of 
the protein to 4M guanidine-HCl causes the protein to unfold with a 
half-life of 1 hour. We would like to compare the unfolding rate with rate 
of dimer dissociation.

We are wondering whether the rate constant for dimer dissociation can be 
obtained from a sedimentation velocity experiment. In one experiment, we 
would determine s-coefficient for monomeric unfolded protein. In the 
second experiment, we would expose the protein to denaturant just prior to 
initiating the sedimentation run, and collect as many scans as possible. 
For data analysis, we would input the s-value for unfolded monomer and try 
to float the s-value for the folded dimer and the rate constant for 
dissociation to obtain the best fit.

Is this approach reasonable? What method should we use to determine s? 
(e.g. the Transport Method may be easiest to modify to include the dimer 
dissociation) Because we are interested in determining the dissociation 
rate constant and not the s-coefficients, can we assume that the diffusion 
constant is same for monomer and dimer? Has anyone attempted this type of 
measurement before?

Best regards,

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Avi Chakrabartty, Ph.D., Associate Professor 
Departments of Medical Biophysics and Biochemistry, 
University of Toronto, Ontario Cancer Institute, 
MaRS Centre, 101 College St., Toronto, Ontario, M5G 1L7, CANADA 
Phone: 416-581-7553 Lab: 416-581-7555 Fax: 416-581-7555
email: chakrab at uhnres.utoronto.ca
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