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<TITLE>[RASMB] difference between experimental and modelled sed coef for protein-DNA complexes</TITLE>
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<DIV> Marcelo,</DIV>
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<DIV>If there is a significant volume change upon formation of the protein-DNA
complex (maybe > ~ 1000 mL/mol of complex), then the vbar of the
complex cannot be calculated accurately using a mass-averaged approach.
We ran into this problem studying the interaction of the UvrD helicase with a
DNA molecule. By sedementation equilibrium methods we measured a
stoichiometric breakpoint that did not agree with the calculated molecular
weight of the protein-DNA complex, and concluded a large, positive volume
change must accompany formation of the complex (Maluf and Lohman,
JMB:325:889, 2003).</DIV>
<DIV> </DIV>
<DIV>N. Karl Maluf</DIV>
<DIV>UCHSC, Denver, CO</DIV>
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<DIV><FONT size=2>-----Original Message----- <BR><B>From:</B>
rasmb-admin@rasmb-email.bbri.org on behalf of Marcelo Nollmann
<BR><B>Sent:</B> Thu 11/20/2003 12:04 PM <BR><B>To:</B>
rasmb@rasmb-email.bbri.org <BR><B>Cc:</B> <BR><B>Subject:</B> [RASMB]
difference between experimental and modelled sed coef for protein-DNA
complexes<BR><BR></FONT></DIV>
<P><FONT
size=2>----------------------------------------------------------------------------------<BR>The
older archived RASMB emails can be found at:<BR><A
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RASMBers,<BR><BR>I am working with a protein-DNA complex for which I did
sedimentation<BR>velocity experiments (interference and absorbance) to
determine its<BR>sedimentation coefficient (using c(s) and finite elements
to calculate<BR><s> from the experimental data). I have done the
experiments a number of<BR>times at different concentrations.<BR><BR>The
buffer in which the complex is contains D2O (43->65%) and
Glycerol<BR>(about 20%). I measured the densities of the buffers and
estimated the<BR>viscosity by using SEDNTERP. However, this last approach
gives the same<BR> viscosities for buffers having different amounts of
D2O (in<BR>disagrement with Matsunaga & Nagashima, J. Phys. Chem. Ref.
Data vol.<BR>12, n. 4, pp. 933-966, 1983.). The vbar I obtained by mass
average<BR>(calculating the predicted vbar for the protein and assuming 0.55
ml/mg<BR>for the DNA).<BR>When I simulate <s>sim, using HYDROPRO or
HYDRO, (with the consensus<BR>model for the structure of this model, based
on x-rays data), I get<BR>quite a big disagreement with the experimental
data (10-20%). By doing<BR>several simulations, and modifying vbar and eta
in reasonble ranges<BR>(.72>vbar>.6, 3cp<eta<4.5cp) I have seen
that <s>sim[w,20](vbar) and<BR><s>exp[w,20](ro,eta,vbar)
terribly depend on these parameters.<BR><BR>Has anyone seen or know of
previous studies where similar disagreements<BR>between calculated and
measured <s> appear for protein-DNA complexes or<BR>similar buffer
conditions??<BR><BR>I would really appreciate any help on this
regard,<BR>Thanks in advace,<BR>Best regards,<BR>Marcelo Nollmann<BR>Glasgow
University<BR><BR><BR><BR>--<BR>I find television very educating. Every time
somebody turns on the set,<BR>I go into the other room and read a
book.<BR>--Groucho
Marx<BR><BR>_______________________________________________<BR>RASMB mailing
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