[RASMB] time to reach equilibrium & protein stability

Fri Oct 1 08:24:00 PDT 2004

Dear RASMBers, 
the protein stability issue is most important in the context of reaching equilibrium. Getting a protein stable in a non physiological environment for many hours or even days is not trivial in many cases, when (auto)proteolytic cleavage or aggregation processes continuously modify molecular mass. 
Fortunately, in this case nature helps AUC, as small proteins which need more time to reach equilibrium are often more stable than large proteins. However, when stability can be improved by addition of glycerol or decreasing temperature, viscosity increases as well and with it the time to reach equilibrium. Optimization is therefore required sometimes. 
Prediction of time to reach equilibrium based on theoretical assumptions is very important to give an initial guess. However, when control profiles show that it takes much longer to reach equilibrium or when equilibrium is never achieved, one should investigate protein stability. Light scattering methods (dynamic and static) turned out very helpful in my lab for doing this and to obtain a first insight into some parameters (average diffusion coefficients or molecular masses) of the system. Such measurements are even faster than Sed velocity experiments... 
Best regards from Basel, 
Hans-Joachim Schönfeld. 
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Dr. Hans-Joachim Schönfeld 
F. Hoffmann-La Roche Inc. 
PRBD-E, B93/5.44 
CH-4070 Basel 
Switzerland 
Tel. (+41) 61 688 28 95 
Fax. (+41) 61 688 90 60 
mailto:hans-j.schoenfeld at roche.com 
-----Original Message-----
From: rasmb-admin at server1.bbri.org [mailto:rasmb-admin at server1.bbri.org] On Behalf Of John Correia
Sent: Wednesday, September 29, 2004 5:31 PM
To: strauss at fmp-berlin.de; rasmb at server1.bbri.org
Subject: Re: [RASMB] time to reach equilibrium

Cheers 
We published a paper in 1977 that predicted the equilibrium distribution from earlier scans: J.J. CORREIA, G.H. Weiss and D.A. Yphantis (1977). "An Extrapolation Method for Reducing Equilibrium Times in Sedimentation Equilibrium Systems," Biophysical J. 20, 153168. The point being, as Holger just mentioned, many samples aggregate with time, or display insolubility, instability. Many of us have observed this with proteins like tubulin which like to associate in polymorphic forms, and with many His tagged proteins. In many of these cases there is a slow loss of material from a "stable" exponential distribution. Information can still be extracted, with caution. Solutions to this problem have included change the pH, ionic strength, etc (obvious), drop the temperature, add reducing agent (TCEP has worked best in limiting cases), remove the His tag, go to a short column format (which speeds up the time dramatically (16-fold for a 4-fold reduction in column height). 
I not only agree with Bo, I insist that you do Sed velocity before you waste time doing sed equilibrium - I suspect most people do this - furthermore for many systems the quantitative analysis must also come from Sed velocity - Sedanal, Sedphat, etc make this very inviting. In our lab sed velocity rules, ie we have no alternative!
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Dr. John J. "Jack" Correia
Department of Biochemistry
University of Mississippi Medical Center
2500 North State Street
Jackson, MS 39216
(601) 984-1522 
fax (601) 984-1501 
email address: jcorreia at biochem.umsmed.edu 
homepage location: http://biochemistry.umc.edu/correia.html
dept homepage location: http://biochemistry.umc.edu/
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