From: Michael Morris <michaelm@pharm.usyd.edu.au>
  To  : rasmb@alpha.bbri.org
  Date: Mon, 27 Nov 2000 11:52:09 +1100

Dear All,

Perhaps some of you would be kind enough to labour through the following and
provide some advise.

We have what is commonly regarded as a worm-like molecule (~60 kDa). It
behaves well in seimentation equilibrium experiments at 20 and 37 C, where
it is strictly monomeric. Rational values for the molecular weight and the
second virial coefficient, B, are returned using Nonlin and the Omega function.

In sedimentation velocity, plots of dc(r)/dt versus s* (where s* is the
apparent sedimentation coefficient) are either obviously asymmetric or
roughly symmetric but broad. If we fit with a single species model, the fits
are either poor or the calculated value of the molecular mass based on s20,w
an D20,w (or the equivalent values having extrapolated to zero
concentration) is about half what it should be because the D20,w is too high. 

OK. All this seems to confirm we have 2 or more monomeric species sedimenting.

When we fit the sed vel data with a model describing two NONinteracting
species, we get good fits, sensible s and D values for the two species, and
the calculated values of molecular mass for both species is 54-60 kDa; i.e.,
pretty well exactly what we expect.

This sounds terrific perhaps but we are not at all convinced that the
monomers in the samples don't interconvert. The reason for this is that we
have done pulsed field-gradient spin-echo (PGSE) NMR experiments. The
results from these experiments unambiguously indicate that (i) there is a
single sedimenting species, or (ii) if there are two or more sedimenting
monomers they must be in rapid equilibrium on the NMR timescale (~20
microseconds!).

Alternative (ii) is the obvious possibility but still does not square with
the sed vel results where good fits and highly sensible returned values of
s, D, and molecular mass are returned on the basis of having two
NONinteracting species.

Is it just coincidence that the sed vel data are fitted so well by the
noninteracting model and return such sensible values? My strong bias is that
the protein adopts a continuum of monomeric conformations which do, in fact,
rapidly interconvert (as suggested by the NMR data). Do models describing
such a situation exist for fitting sed vel data?

With thanks, Michael
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Michael Morris
Senior Lecturer
Pharmaceutical Chemistry	Tel:	+ 61-2-9351-2359
Faculty of Pharmacy A15	  	Fax:	+ 61-2-9351-4391
University of Sydney 2006	Email:	michaelm@pharm.usyd.edu.au 
Australia			
__________________________________________________________________