[RASMB] pressure induced dissociation

Susanne Saalau-Bethell S.Bethell at astex-therapeutics.com
Fri Jul 30 04:49:15 PDT 2010


Hi,

On a slightly different note, I am currently working on a monomeric
protein that has the tendency to aggregate/precipitate  during the final
concentration (Vivaspin centrifugal concentrators) after the final gel
filtration column.  Once I get it to 10mg/ml, I routinely
ultracentrifuge (75000rpm on a TLA110 at 4C) to remove dust, aggregates
etc.  The first time, I expected a large white precipitate pellet at the
bottom of the tube, however I found that the protein had gone back into
solution and was as active as any prep that had not gone through the
precipitation step. Oh happy time!  

Sue 

-----Original Message-----
From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org]
On Behalf Of Tom Laue
Sent: 29 July 2010 19:01
Cc: rasmb at server1.bbri.org
Subject: Re: [RASMB] pressure induced dissociation

Hi-
The pressure dependence of association is through the dV term. For a
monomer-dimer equilibrium, dV is small enough to have little effect on
Ka (the pressure in the cell goes from 1 atm to ~200 atm at 60,000 rpm).

For polymeric assembly, like myosin can undergo, the dV will be larger
simply because the number of subunits undergoing assembly is larger. So,
in general, the number of subunits in an association is important.
Best wishes,
Tom

Arthur Rowe wrote:
> Hi Allen (& all)
>
> Yes indeed - there was this story that a (reversible) myosin dimer 
> existed. Trouble is, it was never found by electron microscopy, and 
> detailed hydrodynamic analysis (Emes & Rowe, 1978 & Sara 
> Suchet-Derechin - lost reference) equally failed to find anything 
> other than monomer, over the (rather low) relevant range in c. Plus, 
> the whole concept sat uneasily with the (later identified) 3-fold 
> rotational symmetry of the native myosin filament. Nice to revisit the

> historic stuff!
>
> But of course this is not to deny the simple fact that hydrostatic 
> pressures of the order of 100+ bar are able to cause dissociation of 
> oligomeric structures. People such as Mike Geeves of U of Canterbury -

> good to meet Mike a couple of weeks back at a Muscle Club Reunion Day
> - have long explored this effect in some detail. Google brings down 
> loads. Synthetic myosin /filaments/ show this effect in the AUC 
> (another Emes & Rowe paper, 1978).
>
> Even some monomeric proteins have been shown to change s values* 
> slightly (Errington _et al_ 2001).
>
> ABW to everyone
>
> Arthur
>
> *fully corrected for all other effects, including solvent density & 
> viscosity changes with pressure
>
> Emes C H & Rowe A J (1978) BBA 537 125-144 Emes C H & Rowe A J (1978) 
> BBA 537 110-124 N Errington, P Mistry & A J Rowe (2001) "Protein 
> hydration varies with protein crowding and with applied pressure: a 
> sedimentation velocity study" Progr. Coll Polym Sci *119 *58-63
>
>
> On Jul 28, 2010, at 21:46, Allen Minton wrote:
>
>     For those of you interested in ancient history, have a look at
this:
>
>
>     An unusual pressure dependence for a reversibly associating
>     protein system; sedimentation studies on myosin.
>
>     Josephs R, Harrington WF.
>
>     Proc Natl Acad Sci U S A. 1967 Oct;58(4):1587-94.
>
>
>     What goes around, comes around.
>
>
>         At 10:58 AM 7/28/2010, smcbryan wrote:
>
>             All,
>             Would anyone/everyone care to comment on these results:
>             
> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC389292/pdf/pnas00083-0121.
> pdf
>
>             and perhaps generalize on how/if they present a common or
>             uncommon
>             phenomenon in centrifugation.
>             Is this something that we should look out for, or is it
>             very specific to
>             this particular complex?
>             thanks,
>             steve
>
>             -- 
>             Steven McBryant, PhD
>             Director
>             Protein Production and Characterization Facility
>             Research Scientist/Scholar
>             Colorado State University
>             970-491-5586
>             _______________________________________________
>             RASMB mailing list
>             RASMB at rasmb.bbri.org
>             
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>
>
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> **********************************************************************
> *********
>
> Arthur J Rowe
> Professor of Biomolecular Technology / Director NCMH Business Centre 
> School of Biosciences University of Nottingham Sutton Bonington Leics 
> LE12 5RD
>
> TEL: 0115 9516156
> FAX: 0115 0516157
> **********************************************************************
> *********
>
>
> ----------------------------------------------------------------------
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>
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--
Department of Biochemistry and Molecular Biology University of New
Hampshire Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
www.bitc.unh.edu
www.camis.unh.edu

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