[RASMB] dimer dissociation kinetics via sed. velocity

[Arthur Rowe]

Greetings Avi - and everyone else

Batch LS does indeed sound to be a feasible approach - at least provided
that you do not get bits of aggregate formed whose 'glow' would mask the
signal you want to analyse.

However, there may be a simple AUC approach which would get round the very
real problems identified by colleagues (especially the problem of the time
to temperature and velocity stability being longer than the time constants
of interest). 

If you use band/zone centrifugation, with your dimeric protein in the
loading compartment and your column filled with 4M-GHCl, then the protein
does not 'see' the dissociating solvent until you are within a minute or so
of selected speed. You then accumulate scans like crazy - must be
interference optics - and using SEDFIT for analysis of multiple short
regions with the Analytical Zone Centrifugation option selected as your
model, you can find the s(w) value as a function of time. From which it is
trivial to find the fraction of monomer present at each 'time' value (i.e.
to be exact about it, at the mid-point of a sequential set of short time
intervals).

Very few people do this method (Analytical Zone Centrifugation). But it's
ultra-economical on sample, easy to carry out, and simple to analyse. Or so
I have always found.

Happy New Year to you and everyone on RASMB!

Arthur


John, Avi & other RASMBers -

Any hydrodynamic measurement is inevitably going to involve
contributions from both unfolding and dissociation.  Discriminating
between the two processes that may be occurring on comparable time
scales is problematic to say the least.  I suggested to Avi that
static LS would be the method of choice, since it is sensitive only
to dissociation.  A batch LS measurement directly gives the
dependence of wt-av MW on time, which is a simple function of the
first order dissociation constant.  Conversely, failure to model the
observed dependence of wt-av MW on time by a simple first-order
dissociation would also be a sensitive flag that something more
complex (and perhaps more interesting) is going on

"If your only tool is a hammer, all problems begin to resemble
nails."   (Who said it first?)  I think this epigram concisely
describes a mindset that afflicts too many in the RASMB
community.   AUC is a lovely technique, but not necessarily the best
way to study everything.

Allen



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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