[RASMB] pressure induced dissociation

Fri Jul 30 05:39:54 PDT 2010

Hi Sue

What a lovely finding. Only wish it happened with more proteins!

Arthur

On Jul 30, 2010, at 12:49, Susanne Saalau-Bethell wrote:

> 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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>
> --
> 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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************************************************************************ 
*******
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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