[RASMB] sedfit vs SOMO vs structure

Fri Jul 8 22:41:01 PDT 2011

Susuma

Agreed - this one must be a monomer. But I must point out that  
estimating weak (Kd > 0.1 mM) is not "quite difficult'' (because of  
non-ideality terms). Simply fitting the inverse of the conventional  
equilibrium (the INVEQ algorithm) is simple and can work up to Kd = 50  
mM (depending on M value of course). See my recent review, below.

Best wishes to you and all

Arthur

Rowe, Arthur J (2011) Ultra-weak reversible protein-protein  
interactions. Methods 54 157-166

On Jul 9, 2011, at 05:02, Susumu UCHIYAMA wrote:

> Mark,
>
> I have a lot of experience that the crystal structure of a protein is  
> dimer but it is monomer in
> solution.
> The Kd of monomer-dimer equilibrium sometimes come close to hundreds  
> of micro-molar or mM , in such
> case it's quite difficult to show dimer presence correctly even in  
> AUC-SE because we have to
> increase concentration of protein, where non-ideal effect become  
> dominant.
> In case of AUC-SV, concentration dependence of s-value in C(s) is also  
> very small.
>
> I think that your protein is monomer in solution, because s-value from  
> SOMO generally quite agree
> with that derived from AUC-SV.
> I agree with John about double minimum is surprising and should be  
> solved by checking sedfit procedure.
>
> Susumu
>
>
>
> (2011/07/09 0:22), John Philo wrote:
>> Mark,
>>
>> I agree with both Arthur and Ewa that either SEC-MALS or a  
>> sedimentation
>> equilibrium experiment would settle this quickly, and I too have seen
>> proteins that are dimeric in crystals but monomeric in solution.
>>
>> But regarding the existing velocity data, what you haven't said is  
>> what
>> happens as a function of concentration. For any new molecule it is  
>> really
>> essential for proper interpretation of velocity data to do a dilution
>> series. It may well be that you have a weak rapid monomer-dimer  
>> association
>> and your 2.60 S c(s) "species" represents a dynamic mixture with a  
>> small
>> amount of dimer.
>>
>> A further point is that c(s) is really not the best way to determine  
>> a MW
>> from velocity data. If the c(s) says you have only a few species,  
>> then you
>> should fit the data by an explicit model with that number of species  
>> (in
>> SEDFIT the 'non-interacting discrete species' model) to directly get  
>> the
>> mass and sedimentation coefficient of your main species. This will  
>> also
>> allow you to determine the confidence limits for those quantities,  
>> which you
>> cannot do with c(s).
>>
>> It also seems rather surprising to me that you find a double minimum  
>> for
>> f/f0 in the c(s) fits. If you are fitting the meniscus and/or cell  
>> base
>> positions, you should probably check whether there is possibly  
>> something odd
>> (non-physical) going on with those.
>>
>> John
>>
>> -----Original Message-----
>> From: rasmb-bounces at rasmb.bbri.org  
>> [mailto:rasmb-bounces at rasmb.bbri.org] On
>> Behalf Of Arthur Rowe
>> Sent: Friday, July 08, 2011 7:38 AM
>> To: Ewa Folta-Stogniew
>> Cc: rasmb at server1.bbri.org
>> Subject: Re: [RASMB] sedfit vs SOMO vs structure
>>
>> 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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************************************************************************ 
*******
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

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