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

[Karl.Maluf at UCHSC.edu]

          Marcelo,

	 
	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).
	 
	N. Karl Maluf
	UCHSC, Denver, CO

		-----Original Message----- 
		From: rasmb-admin at rasmb-email.bbri.org on behalf of Marcelo Nollmann 
		Sent: Thu 11/20/2003 12:04 PM 
		To: rasmb at rasmb-email.bbri.org 
		Cc: 
		Subject: [RASMB] difference between experimental and modelled sed coef for protein-DNA complexes
		
		

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		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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