[RASMB] Small peptides and AUC

[Jo Butler]

Dear All,

On v-bar Dave raises a very valid point.  There is however one major 
problem with trying to match densities with the solvent and this is that 
lipids and detergents usually do not have the same v-bar themselves, so 
it is not possible to match both in the same experiment.  This has often 
been overlooked in density-matching experiments.

If I may push a personal opinion, measuring the density increment 
directly has great advantages and is the one really reliable method.  We 
discussed the whole problem in  Butler, P. J. G. & Tate, C. G. (2005) 
Correcting for the Buoyancy of Macromolecules;  Density Increments and 
Apparent Partial Specific Volumes with particular reference to the study 
of membrane proteins in Analytical Ultracentrifugation:  Techniques and 
Methods (Scott, D., Harding, S. & Rowe, A., eds) pp. 133-151, Royal 
Society of Chemistry, London.

Yours,

Jo


Scott David wrote:
>
> Hello All,
> Following on from discussions of 90 % DMSO,etc. There are more 
> applications (at least through the NCMH) to look at peptides in 
> membranes and detergents, and talking to people in the AUC field, this 
> is a somewhat growing field as people become less fearful of 
> membrane-bound stuff. At this juncture I think it would be good if we 
> could start a discussion strand of what to watch out for when 
> analysing these samples. Original references very welcome.
>
> Here's several very obvious things to get us started.
>
> 1) vbar. The bouyancy of the peptide changes with binding of lipids 
> and detergent. This has to be either (a) matched out by having a 
> solvent that has the same density as the lipid/detergent or (b) 
> calculated from the density increment. I also had a referee once ask 
> me years ago whether I had corrected for charge on a small peptide. 
> The answer was "no" as the peptide was actually a reasonably sized 
> protein, but the original reference for this would be good.
>
> 2) Speed. Peptides are small, you will need probably the highest speed 
> possible to analyse them, and for the XL-A/I that's 60K and the 
> associated problem of buying the correct centrepieces (Aluminium or 
> SpinAnalyticals SedVel60K).
>
> 3) Co-sedimentation of the solvent. Peptides are small, therefore have 
> low S values. I seem to recall that Na^+ sediments at around 0.3 S, 
> therefore you will need to think about how the sedimentation of your 
> solvent components affect the sedimentation of your sample. Not 
> trivial if these affect the solubility and stability of your peptide.
>
> 4) Small S values. Are our algorithms good enough?
>
>
> Enjoy!
>
> Dave.
>
>
>
> Dr. David J. Scott
> Lecturer in Physical Biochemistry
> National Centre for Macromolecular Hydrodynamics
> School of Biosciences
> University of Nottingham
> Sutton Bonington Campus
> Sutton Bonington
> Leicestershire
> LE12 5RD
> United Kingdom
> dj.scott at nottingham.ac.uk
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-- 
Dr P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 0QH, UK.
Tel. +44 (0)1223 402296