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

[Peter Schuck]

Gwen,
since you suspect the presence of larger oligomers, and also a 
time-dependent aggregation, I would suggest to try sedimentation velocity.

You should be able to identify if there's large oligomers present, and 
resolve them easily, e.g., by c(s) .  Although the peaks in the 
sedimentation coefficient distribution wouldn't represent the s-values of 
those species participating in the fast reversible equilibrium (e.g., your 
monomer and dimer or larger oligomer), any large irreversible aggregates 
should be clearly separated.  Also, from the range of s-values you might be 
able to get clues about the oligomers formed.  Integration of the c(s) 
distribution over the range of s-values of the associating species can give 
an isotherm of weight-average s versus concentration, from which you may be 
able to deduce an association scheme and get a binding constant.  If the 
system is well-behaved and aggregates don't show up at the shorter 
experimental times of sedimentation velocity, a direct boundary modeling 
analysis may also be possible.  Important would be to have a very high 
rotor speed, and a very large concentration range (perhaps by varying the 
monochromator wavelength).

Of course this doesn't always work, there can sometimes be pressure induced 
dissociation, or it may not always be possible to unambiguously define the 
association scheme, either.  However, in my hands it always gave a lot of 
additional information, because it provides much better resolution of 
species.  This may at least help to develop the right model for 
interpreting the sedimentation equilibrium.

Peter



>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