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

[John Correia]

In  most versions of Nonlin (I though this was also so for the Beckman
version) you can change the K's to be either global or independent
meaning a different K for each channel of data - under fitting look for
common K's - if within error they are the same K value then the data is
well behaved, ie reversible.  I usually just ceheck the SD to see if its
about the same as the rror bars on a global K value.  If K2 varies
outside that SD then you have heterogeneity or the wrong model.  Usually
heterogeneity or aggregation gives decreasing K values with increasing
loads.  An increasing K means more asociation is happening with
increasing conc and thus probably higher order spoecies are being formed
- add additonal terms K's for larger N values.



>>> "Yun-Ru (Ruby) Chen" <ychen5 at unity.ncsu.edu> 06/05/02 10:27AM >>>
Dr. Correia:
Thanks for your help. I have sent out a lot more information through
the
system. You will see it soon.
But I am not sure what do you mean by making K2 independent. Does that
mean
fixing K2 to a certain guess and have others varying? By fitting the
data to
a dimer to octamer equilibrium, I got "K2" increasing with increasing
loading concentration. Please let me know.
Sincerely,
Ruby

----- Original Message -----
From: "John Correia" <jcorreia at biochem.umsmed.edu>
To: <rasmb at rasmb-email.bbri.org>; <ychen5 at unity.ncsu.edu>
Sent: Tuesday, June 04, 2002 2:17 PM
Subject: Re: [RASMB] Fw: Can 0.25M of NaCl take care of
nonidealbehavior of
a protein?


> 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 
> -------------------------------------------------------------------
>
>
>
>
> >>> "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
>