[RASMB] dimer dissociation kinetics via sed. velocity

[Dror Noy]

Avi,
Check out Peter Schuck's manuscript in Biophysical Journal 2005, p.  
619. As far as I understand, it is possible to extract kinetic  
information for Koff rates between 0.01 to 0.00001 s-1 using  
sedimentation velocity. The analysis protocol is implemented in  
Peter's sedphat program, and for sure he can provide the detailed  
answer to your question.
best,
Dror

On Jan 9, 2007, at 10:55 PM, Avi Chakrabartty wrote:

> 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,
>
> -------------------------------------------
> 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
> --------------------------------------------
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb



=========================
Dror Noy, Ph.D.
Plant Sciences Dept.
Weizmann Institute of Science
Rehovot 76100
Israel

e-mail: dror.noy at weizmann.ac.il
Tel: (972)-8-934 4300
Fax: (972)-8-934 4184