[RASMB] sedfit vs SOMO vs structure

[Arthur Rowe]

Hi Mark

The energetics of monomer-monomer interaction in the crystalline state  
differ greatly from those applicable in solution. The fact that you see  
as dimer by XRC is not hard evidence for the presence of a dimer in  
solution. I recall working in the past on CAT (chloramphenicol  
acetyltransferase) which we found to be trimeric in solution under a  
range of solvent conditions: yet by XRC in the presence of Mn two of  
these trimeric units were clearly associated to form a hexameric  
structure within the unit cell. So I don't think that you have any real  
evidence from XRC to support the presence of dimer in solution.

It's fascinating that you can get two minimal positions by fitting with  
SEDFIT, but I cannot believe that the fact that initialising the f/f0  
value close to 2.3 give you a credible dimer value via c(M). But all  
this shows I think is the danger of letting curve-fitting numerology  
overwhelm common sense. If your dimer has so large an f/f0, then your  
monomer must be either hugely swollen, immensely assymmetrical, or  
both. I guess that inspection of the monomer structure within the XRC  
structure shows nothing of the sort?

SEC column MW's are not credible, for well-known reasons. And I don;t  
agree with Ewa that having DLS on-line would totally resolve the issue,  
since SEC-DLS remains a non-absolute method. SEC-MALLS of course would  
be excellent - if you have the kit. But you do have an AUC, and doing a  
simple overnight SE run and analysing by even the simplest software  
approach would immediately tell you, on firm theoretical grounds,  
whether you had a 30 kDa or a 60 kDa entity.

Regards to you, and to all

Arthur

************************************************************************ 
*******
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

************************************************************************ 
*******

On Jul 8, 2011, at 13:54, Ewa Folta-Stogniew wrote:

> At 06:08 AM 7/8/2011, Mark Agacan wrote:
>
>> Hello RASMB,
>> Sedfit c(s) analysis of a protein I'm working with shows a 30 kDa  
>> peak with S = 2.60 and f/f0 of 1.2, that corresponds to monomer.
>> SOMO in Ultrascan using the pdb file for this protein predicts a 30  
>> kDa monomer at 2.42, with f/f0 of 1.19.  It's good agreement with the  
>> sedimentation velocity results.
>> However, the crystal structure, SEC, etc. indicate this protein  
>> should be a dimer.
>> Starting with the default value of 1.2 for the frictional ratio and  
>> floating in sedfit returns a value of 1.2, with MW that corresponds  
>> to monomer.  RMSD = 0.009756
>> Doubling this value to 2.4 and floating gives a value of 2.3, and  
>> assigns a mass that corresponds to dimer to the main peak in the c(s)  
>> analysis.  RMSD = 0.009324
>> Thus there appear to be two energy minima for the frictional ratio,  
>> depending which initial value is used.  The RMSD value is lower when  
>> the higher value of f/f0 is used.
>> Would you, on the basis of RMSD values, assign monomer or dimer to  
>> this peak?
>
> I would run SEC/MALLS with DLS on line- to get direct measurement of  
> MW and Rh. which will allow you to determine f/fo value. .
>
> See attached.
>
> Ewa
>
>
>> Thanks,
>> Mark
>>
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
>> Dr Mark Agacan, Scientific Officer for the Division of Biological  
>> Chemistry and Drug Discovery,
>> Wellcome Trust Biocentre, College of Life Sciences, University of  
>> Dundee, Dundee, DD1 5EH
>> Tel: +44 1382 386095    Fax: +44 1382 345764    Mobile: 07525 451 117
>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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