From: John Philo <jphilo@mailway.com>
  To  : Michael Morris <michaelm@pharm.usyd.edu.au>
  Date: Mon, 27 Nov 2000 08:55:33 -0800

RE: 2 conformations of monomer

Michael,

I think your data is unambiguous that there is some form of conformational
heterogeneity, but it might indeed be coincidence that the apparent
diffusion coefficients from a 2 non-interconverting species model are
consistent with the monomer mass. I think you would have a much stronger
case if you can get the same result from a whole-boundary approach like
SEDFIT or SVEDBERG, where you can use data over the whole span of the
experiment, rather than the limited time span one can use in DCDT analysis.

With regard to consistency with the PGSE experiments, I'm not too familiar
with the method but if I remember correctly with PGSE you are basically
detecting diffusion coefficients. Would you necessarily be able to resolve
two long-lived species with very slightly different diffusion coefficients?
The analytical centrifuge has a tremendous advantage because there is real
physical separation, not just computer algorithmic separation.

Finally, if you really have long-lived conformations with significantly
different hydrodynamics, you would expect to also be able to see this in
native gel electrophoresis (or its CE equivalent). Have you thought of
trying that?

John Philo
Alliance Protein Laboratories
www.ap-lab.com

-----Original Message-----
From: Michael Morris [michaelm@pharm.usyd.edu.au]">mailto:michaelm@pharm.usyd.edu.au]
Sent: Sunday, November 26, 2000 4:52 PM
To: rasmb@alpha.bbri.org
Subject:


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
__________________________________________________________________

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
__________________________________________________________________