From: Joel Mackay <j.mackay@biochem.usyd.edu.au>
  To  : Michael Morris <michaelm@pharm.usyd.edu.au>
  Date: Tue, 28 Nov 2000 09:24:45 +1100

RE: 2 conformations of monomer

>hi Michael,

My contribution is to suggest that the PFG experiments are a bit dodgy 
because of the difficulty in estimating things like the hydration etc.
If you really think that there are some conformational dynamics going on 
(and it appears that you can prepare protein for NMR), you could try 
labelling with 15N and measuring the standard 15N relaxation parameters 
(T1, T2, and 15N-1H NOE) - these give you a residue-specific and fairly 
unambiguous indication of what is going on with the protein backbone.
cheers from over City Rd,
joel

>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
>__________________________________________________________________

*****************************************************************
Dr Joel Mackay
ARC Research Fellow
Department of Biochemistry
University of Sydney,
Sydney, NSW 2006
Australia
ph +61-2-9351-3906
fax +61-2-9351-4726
WWW: http://www.biochem.usyd.edu.au/staff/mackay/
Sydney Protein Group Website:
http://www.biochem.usyd.edu.au/spg/
****************************************************************