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

Jo Butler pjgb at mrc-lmb.cam.ac.uk
Wed Feb 26 04:38:01 PST 2003


Dear Gwen,

While I am not competent to discuss details of fitting with MacNonlin, I 
may be able to help a little by commenting on some work which I did with 
colleagues some time ago (Cabezon, et al. (2000)  J. Biol. Chem. 275, 
25460-25464).  In this we were looking at the inhibitor of the 
mitochondrial F1-ATPase (IF1), and we found that the monomer was 
exclusively a dimer, with absolutely no trace of dissociation - so in 
practice to get any good fitting to the data we needed to consider M1 as 
twice the true value.  At appropriate pH, this dimer further dimerises to 
give an inactive tetramer, which shows little dissociation back to dimer.
Subsequent structural work showed that the protein has a coiled-coil 
structure as a dimer, with these coils coiling together further to give the 
tetramer, and thus fully explained our inability to get monomers at any 
realistic concentration and also the lack of significant dissociation of 
tetramer at the pH where this forms.

In view of your observation that your protein is again a coiled-coil, I 
thought that this experience might be helpful to consider.

Yours,

Jo

--On Tuesday, February 25, 2003 7:48 pm -0700 "Guinevere A. Murphy" 
<Guinevere.Murphy at Colorado.EDU> wrote:

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> 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
> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
> ~~
>
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P.J.G. Butler,
MRC Laboratory of Molecular Biology,
Hills Road, Cambridge, CB2 2QH, UK.
Tel. +44 (0)1223 402296



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