[RASMB] dissociation of multiprotein complex in ultracentrifuge

[John J. Correia]

An interacting system should not run as a complex and free components unless there are slow kinetics or incompetent monomers.  If the affinity is low then you shouldn't see the fully associated complex.  You didn't mention the method but I presume its c(s) which can turn a trailing tail of a reaction boundary into multiple peaks.  Doesn't mean its free components.  To know any of this will confidence you must do a concentration series, changing ratios, and doing direct boundary fitting.

The ribosome story (I presume you mean 30s + 50s to 70s) is complicated by the presence of tight and loose couples, meaning a difference in Mg dependence with one fraction assembling at ~2 mM Mg and another fraction at 12 mM Mg.  This gives rise to three peaks at 30, 50 and 70s.  The 30s is a slower trailing component that you might see with excess component, but most of the 30 and 50s material is actually due to nonreacting loose couples.  This is why I stress you must fit the reaction boundary data to a reversible and or kinetic model.

________________________________
From: rasmb-bounces at rasmb.bbri.org [rasmb-bounces at rasmb.bbri.org] On Behalf Of Mark Agacan [M.Agacan at dundee.ac.uk]
Sent: Tuesday, December 07, 2010 6:03 AM
To: rasmb at rasmb.bbri.org
Subject: [RASMB] dissociation of multiprotein complex in ultracentrifuge

Hello,

Very often when I run a velocity experiment at 45 K rpm with a multiprotein complex, I see peaks with MW values corresponding to the complex, and also to many of the individual components of the complex.  I have read the RASMB archives on the issue of pressure-induced dissociation of sedimenting ribosomes.  Do other people find this is happening for multiprotein complexes, perhaps those that are very weakly associated?

Best Regards,

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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