[RASMB] dimer dissociation kinetics via sed. velocity

[John Philo]

Avi,

I think this is going to be a difficult approach, especially since with a
one hour half-life much of the reaction will happen before you ever get your
sample in the rotor and equilibrated to temperature.

For sure you cannot simply assume the diffusion coefficient is the same for
monomer and dimer. Remember that M, s, and D are linked through the Svedberg
equation---if you fix the same D for monomer and dimer you are
simultaneously saying the sedimentation coefficients for monomer and dimer
differ by exactly a factor of 2. 

If you do try the SV experiment you may want to consider analysis with Walt
Stafford's SEDANAL, where you can write and fit any kinetic model.

Since you are only interested in the kinetics, and given the time scale, why
not try to do this measurement using size-exclusion chromatography ("gel
filtration")? With 4 M guanidine in the elution buffer the unfolded monomer
will hopefully not stick to the column matrix, and with a silica-based
column the separation time should be < 15 min. You could simply vary the
time of unfolding prior to injection.

Depending on the MW of this protein and the concentration, you might be able
to directly measure the kinetics of dissociation using batch-mode light
scattering (in principle either static or dynamic could work). This is also
potentially a good experiment for Allen Minton's LS setup with
computer-driven syringes for mixing.

John

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
Behalf Of Avi Chakrabartty
Sent: Tuesday, January 09, 2007 12:56 PM
To: rasmb at server1.bbri.org
Subject: [RASMB] dimer dissociation kinetics via sed. velocity


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
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