[Fwd: Fwd: RE: [RASMB] dimer dissociation kinetics via sed. velocity]

[Walter Stafford]

 >Date: Wed, 10 Jan 2007 14:04:59 -0500

>To: jphilo at mailway.com, 'Avi Chakrabartty' <chakrab at uhnres.utoronto.ca>, 
>rasmb at server1.bbri.org
>From: Ewa Folta-Stogniew <ef55 at email.med.yale.edu>
>Subject: RE: [RASMB] dimer dissociation kinetics via sed. velocity
>
>Avi,
>
>I would agree with John suggestions about using LS, especially in batch 
>mode and with syringe arrangement as described by Minton.   I would 
>however strongly advocate for using both DLS and static LS as DLS may not 
>be able to differentiate between unfolded monomer and folded dimer since 
>they both would presumably have greater Rh than a folded monomer.  Static 
>LS on the other hand, should determine weight-average molar mass, from 
>which one can compute the ratio of monomer and dimer (if these are the 
>only two species present).   My major fear would be formation of 
>aggregates from the unfolded monomer, which would overwhelmed LS signal.
>
>I would combine John suggestions, and go for SEC/LS in 4M GuHCl.   The 
>timing can be easily monitored and the time scale is perfect for SEC on a 
>small sturdy column that can be run fast (i.e. 1 ml/min), the only problem 
>is the dilution on SEC, which would affect the dimerization status.  If I 
>were to run such experiment, I would go for a zone SEC on a small, home 
>build SEC column, in which the aggregates can be separated from the lower 
>MW species (accounted for and characterized) and the plateau peak would 
>contain the mixture of monomer and dimer at any given time point and the 
>distribution can be computed from the average MW.
>
>Ewa
>
>At 12:40 PM 1/10/2007, John Philo wrote:
>>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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>>
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Walter F. Stafford
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