[RASMB] difference between experimental and modelled sed coef for protein-DNA complexes

[Marcelo Nollmann]

Dear RASMBers,

I am working with a protein-DNA complex for which I did sedimentation 
velocity experiments (interference and absorbance) to determine its 
sedimentation coefficient (using c(s) and finite elements to calculate 
<s> from the experimental data). I have done the experiments a number of 
times at different concentrations.

The buffer  in which the complex is contains D2O (43->65%) and Glycerol 
(about 20%). I measured the densities of the buffers and estimated the 
viscosity by using SEDNTERP. However, this last approach gives the same 
  viscosities for buffers having different amounts of D2O (in 
disagrement with Matsunaga & Nagashima, J. Phys. Chem. Ref. Data vol. 
12, n. 4, pp. 933-966, 1983.). The vbar I obtained by mass average 
(calculating the predicted vbar for the protein and assuming 0.55 ml/mg 
for the DNA).
When I simulate <s>sim, using HYDROPRO or HYDRO, (with the consensus 
model for the structure of this model, based on x-rays data), I get 
quite a big disagreement with the experimental data (10-20%). By doing 
several simulations, and modifying vbar and eta in reasonble ranges 
(.72>vbar>.6, 3cp<eta<4.5cp) I have seen that <s>sim[w,20](vbar) and 
<s>exp[w,20](ro,eta,vbar) terribly depend on these parameters.

Has anyone seen or know of previous studies where similar disagreements 
between calculated and measured <s> appear for protein-DNA complexes or 
similar buffer conditions??

I would really appreciate any help on this regard,
Thanks in advace,
Best regards,
Marcelo Nollmann
Glasgow University



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