[RASMB] Help! No model fits my sedimentation eq data

John Philo jphilo at mailway.com
Wed Feb 26 11:41:01 PST 2003


I certainly agree with all the earlier comments, but one additional thing
that may simplify your situation would be simply to drop the loading
concentrations. You've already stated that even your lowest concentration
sample seems to be nearly all dimer. Your numbers also indicate that your
highest concentration sample is a loading concentration of 2.3 mg/ml. At
25000 rpm a quick check with the Beckman equilibrium data simulator
indicates you are probably reaching concentrations over 10 mg/ml at the base
of the cell (and >20 mg/ml at 35K), which means you are well into a range
where low affinity associations ("aggregation") are very likely and
non-ideality will also make analysis very difficult. 

If you really have a specific coiled-coil it seems to me you shouldn't need
nearly such high concentrations, and further if your main focus is to get a
reasonable estimate for the monomer-dimer Kd then you may be able to do that
by staying away from the concentration regime where higher order species
become significant. 

Why not drop the concentration, perhaps using 230 nm instead of 280, and see
if things make more sense?

John Philo
Alliance Protein Laboratories

-----Original Message-----
From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of Guinevere A. Murphy
Sent: Tuesday, February 25, 2003 6:49 PM
To: rasmb at rasmb-email.bbri.org
Subject: [RASMB] Help! No model fits my sedimentation eq data


----------------------------------------------------------------------------
------
The older archived RASMB emails can be found at:
http://rasmb-email.bbri.org/rasmb_archives
and current archives at http://rasmb-email.bbri.org/pipermail/rasmb/
Search All the Archives at: http://rasmb-email.bbri.org/rasmb_search.html
----------------------------------------------------------------------------
------


Dear RASMB,

I am (a novice at) working with equilibrium sedimentation data for a 16.9
kDa protein (E280=2680 /M cm), collected at three concentrations of 56, 94
and 136 microMolar. I collected data at four speeds, 20, 25, 30 and 35 krpm.
The protein is a long dimeric coiled coil, so I expect to have a
monomer-dimer equilibrium. There is a possibility of higher order structure,
so a mono-dimer-tetramer equilibrium would not be out of the question,
either.

I have been analyzing the data using MacNonlin. For the two 20K data sets I
collected, I can locally fit the lowest 56 micromolar data to a single ideal
component model, (initial guesses: sigma=1.8 (dimer), deltaY=3E-3, and
lnA=-2.6) I get nice random residuals, and a sqrt variance of ~4E-3. The
fitted sigma corresponds to a 29 kDa component (between the expected 16.9
and 33.8 monomer and dimer MWs).

If I fit to a monomer-dimer equilibrium for this data set, I also get
reasonable sqrt variances (4E-3) and nice random-looking residuals, and I
get a Kd of 28 microMolar (using the 1/Ka, after converting the Ka from
absorbance units with the formula Ka(M)=[Ka(AU)*E(monomer)*(1.2cm path)]/2).
I view this high number with skepticism, both because it is pretty high, for
what I might expect, and because I am only fitting one concentration and
speed.

So, I would like to add in other data sets, however I find that I am unable
to fit any of the other data to a reasonable model in a manner which
produces anything like random residuals, and the sqrt variances are all high
(well above 1E-2). This is true for fitting to single ideal component,
monomer-dimer, monomer-trimer, dimer-tetramer and many others that are not
as obvious.

There are two observations regarding local fitting of one of the data sets
that I am having trouble with that I think might be suggesting nonspecific
aggregation of the protein at higher concentrations, and later time points
(20K was collected first):

a.) If I vary B, the second virial coefficient, I do get a random
distribution of residuals, and sqrt variance of 3E-3, but the value B gets
fitted to is -0.57. I know positive values of B suggest nonideality, but
does negative mean it is ideal and just isn't being fitted to the proper
model?

b.) If I set sigma=1.9 (for dimer) and don't vary it, and then vary N2(init
guess at 2, so init guess is for dimer-tetramer equilibrium) lnK2 (init
guess at 8), lnA (init guess -3), delY (init guess 7E-2), then I get the
most reasonable fit to the data I've seen, and the residuals are not totally
random, but more dispersed and random than any other model, with a sqrt
variance of 6E-3.  However, the fitted parameters go to N2=17.5, and
lnK2=9.5!

This result suggests to me that either I've got one heck of a large
oligomer, or there is nonspecific aggregation occurring.

Would anyone have insight into this this difficult-to-fit data? And if it is
likely nonspecific aggregation, is there a definitive diagnostic that would
show that with the data I already have?

Thanks very much for taking the time to read this, and for your suggestions
and comments!

Gwen Murphy

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Guinevere A. Murphy		    Chemistry and Biochemistry Department
murphyg at ucsu.colorado.edu	    The University of Colorado at Boulder
http://ucsu.colorado.edu/~murphyg
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

_______________________________________________
RASMB mailing list
RASMB at rasmb-email.bbri.org
http://rasmb-email.bbri.org/mailman/listinfo/rasmb




More information about the RASMB mailing list