[RASMB] Fw: Can 0.25M of NaCl take care of nonideal behavior of a protein?

[John Correia]

If you are fitting to a monomer-dimer model? what happens when you make
K2 independent - do the channels fit separately with random residuals
and do the K2 values decrease with increasing loading concentration
which would indicate heterogeneity or aggregation.  Reducing agents will
not fix S-S linkage problems that originate with the tendancy of a
protein to aggregate.  Reduction will simply make SH groups available to
crosslink to other monomers/dimers and typically makes the aggregation
worse.  TCEP is a stronger reductant and for reversible S-S problems
will fix the aggregation, but likewise it will not fix the tendancy to
aggregate independent of S-S.

John has asked you for concentration ranges to interpret the
nonideality suggestion, but also tell us the range of OD's you are
loading and fitting.  It would also be helpful to know what speed/sigma
values are you running.




-------------------------------------------------------------------
 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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>>> "Yun-Ru (Ruby) Chen" <ychen5 at unity.ncsu.edu> 06/04/02 11:02AM >>>

----- Original Message ----- 
From: Yun-Ru (Ruby) Chen 
To: rasmb at rasmb-email.bbri.org 
Sent: Friday, May 31, 2002 2:37 PM
Subject: Can 0.25M of NaCl take care of nonideal behavior of a protein?



Dear RASMB members:
              I have currently run into problems by analyzing my data
from sedimentation equilibrium studies. I am very confused and
frustrated about it.
I hope if any of you can give me some ideas. I am working on a 10KDa
protein domain which is suppose to be a helical bundle. The pI of the
protein is calculated to be about -1.5 at pH 8. I have ran the samples
in two different buffers, one in 30 mM Tris, pH8, and one in Tris with
250 mM NaCl. Both buffers contain 0.2mM DTT and are at pH 8. However, we
cannot fit all the data to a single assembly model. I have tried to put
in different guesses of "B" values for nonideality, but the fits are
even worse. Moreover, the data from the salt buffers look better then
the one without. Therefore, does that mean 0.25M NaCl is not enough or
there is something else that I should be aware of? My protein is much
less soluble in the presence of NaCl, so it is a bit hard for me to
increase to salt concentrations.
Thanks very much if you can share your idea with me.

Sincerely, 
Ruby

Yun-Ru (Ruby) Chen
Ph.D. candidate
Department of Structural and Molecular Biochemistry
North Carolina State University