[RASMB] disulfides

[Peter Schuck]

Hi Holger,
You can use SEDPHAT to analyze monomer-dimer (or other self-association) 
equilibria incorporating incompetent monomer or dimer.  The models in 
SEDPHAT are for both sedimentation velocity and sedimentation equilibrium, 
and I would use the latter one with mass balance conservation.  You can 
assess the reliability of the incompetent fraction simply by statistical 
means - just compare the chi-square of the fit that you get without 
considering incompetent fractions, or if you fix the fraction at 
non-optimal values. From sedimentation equilibrium, I'd expect the number 
to be reasonably reliable if you have many data sets over a  large 
concentration range and different rotor speeds.
Peter



At 03:32 PM 6/4/03 +0200, you wrote:
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>Hello all,
>
>there is the following situation: a given protein, 20 kDa, recombinantly
>expressed in e.coli, 5 free cysteines, one band in SDS-PAGE/Coomassie (no
>silver stain done), 2 species in the MALDI-spec, monomer and covalent
>dimer, nice CD and melting curve. The buffer is Tris pH 7.4, 100 mM NaCl,
>5 mM beta-Mercapto.
>
>OK, there seems to be a portion of specific disulfide linkage, and the
>system slighlty self-associates (point-average Mw, integral
>c(S)/concentration all increase), but it's close-to-impossible to describe
>the equilibrium gradients by a simple standard model, which is due, I
>think, to a significant contribution of the covalent linked dimer to the
>measured concentration distribution.
>
>So, here are my questions:
>
>- if one could measure the fraction of covalent dimer, one could simulate
>its equilibrium distribution, and substract it from the experimental
>gradients; how would one measure this precisely?
>
>- if it can't be measured precisely, maybe one could fit directly for the
>fraction of covalent dimer? (which programm would allow you to do this?
>How to judge the reliability of this number?)
>
>- (that's more general): what does the term "incompetent monomer" mean on
>a structural basis (assuming that folded equals competent)? (Treating such
>a thing would be very much the same as with a covalent dimer, I presume?)
>
>Greetings to all, Holger
>
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>
>Holger Strauss
>
>Forschungsinstitut fuer Molekulare Pharmakologie (FMP)
>Robert-Roessle Strasse 10
>
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>
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>Science is spectrum analysis; art is photosynthesis.
>
>                                                     Karl Kraus
>
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