From: Walter Stafford <stafford@bbri.org>
  To  : Les Hicks <hicks@ualberta.ca>
  Date: Sat, 14 Jul 2001 19:25:04 -0400

Re: Differentiating between folded and unfolded states in sed vel

Hi Less,
	At @ 7 kDa you probably wouldn't see any boundary because of 
diffusion. But theoretically if the rate of interconversion between 
the conformational isomers were fast compared to the time scale of 
sedimentation, you would see a single boundary with values of s and D 
that would give you correct molecular weight.  Proteins are always 
"breathing" rapidly anyway, so this is actually the most common 
situation. The peak will start to become broader only if the isomers 
have lifetimes on the order of 60-100 seconds or larger.

So to answer your question, you could only resolve the boundary into 
two isomers if the lifetimes were longer than 10^4 to 10^5 seconds. 
Anything between about 100 and 10^4 seconds would be a smear that 
could only be analyzed by curve fitting an appropriate kinetically 
controlled isomerization model to the data.

Walter Stafford


At 15:57 -0600 7/13/01, Les Hicks wrote:
>Hi folks,
>
>     Here's a hypothetical question.  Say you have an approximately 7 kDa
>protein that is known from other techniques to undergo reversible folding /
>unfolding in phosphate buffer.  If the rate of folding / unfolding is fast
>(within the time course of a sed vel run, would it be possible to
>differentiate between the two states from the sed vel data?  I would think
>all you would see is a slightly broader boundary and that you would not be
>able to identify two individual boundaries for the two states.  Any takers
>on this question?  In any event, have a good week-end.
>
>Regards,
>
>Les Hicks

-- 
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Walter F. Stafford III, Ph.D.
Senior Scientist
Analytical Ultracentrifugation Research Laboratory
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA  02472-2829
and
Associate in Neurology
Massachusetts General Hospital
Harvard Medical School
Boston MA 02115

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tel: (617) 658-7808
fax: (617) 972-1753

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