I am curious why you are so exercised about the oligomerization state.
Is the tetramer elongated or close-to-spherical in the crystal, i.e.,
maybe its shape is perturbing its SEC mobility. Right now, it seems
that you have LS and SEC saying that the thing is at least oligomeric
in solution--why not try a quick cross-linking experiment with
gluteraldehyde, or a longer experiment engineering in cysteines to try
to cross-link the crystallographic tetramer?
Also, maybe something in the crystallization buffer perturbs the
oligomerization state?
Also, what is your reasoning for saying that PEG conditions favor the
correct oligomeric state?
Jacob
On Thu, Jun 2, 2011 at 2:38 PM, Kushol Gupta <[log in to unmask]> wrote:
> Hi Chad,
>
>
>
> Re SV: the way I understand it, there’s a pitfall to mass determination in
> the case of interacting systems (including oligomers) – the accurate
> determination of the S value for a complex is going to be affected by its on
> and off rate and the concentration at which it is analyzed (and hence, any
> derivation of mass?). From what I understand from one resource, even
> 10-fold above the Kd for a rapid association, the highest peak in a c(S)
> distribution may not be the actual complex S value. No doubt, though, a
> well-behaved single species should provide a good determination of mass.
>
>
>
> Kushol
>
>
>
> Kushol Gupta, Ph.D.
>
> Research Associate
>
> Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine
>
> [log in to unmask]
>
> 215-573-7260 / 267-259-0082
>
>
>
> From: Chad Brautigam [mailto:[log in to unmask]]
> Sent: Thursday, June 02, 2011 2:38 PM
> To: Kushol Gupta
> Cc: [log in to unmask]
>
> Subject: Re: [ccp4bb] larger molecular weight shown by analytic
> ultracentrifugation
>
>
>
> Dear Kushol & Jerry,
>
> I have to take exception to Kushol's contention about SV. As long as the
> buffer and protein parameters are correct and the sample is well behaved
> (i.e. not undergoing dynamic rearrangement on the time scale of the SV
> experiment, not aggregated, homogeneous), one can derive very accurate molar
> masses from SV, and it is far superior in this respect than SEC.
>
> However, as you aptly point out, the problem may lie in the sample's
> behavior. If the protein is populating multiple oligomeric states, or if it
> is undergoing a fast interconversion of such states, or if it is not pure,
> spurious masses may be calculated. If such pathologies are observed, it's
> not clear to me that a sedimentation equilibrium experiment would help. For
> a complicated interaction model, SE data (and AUC data in general) can be
> difficult to analyze, and some model assumptions are almost always present.
>
> So, the questions are:
>
> 1. Did Jerry run SV or SE?
> 2. If the former, then did he see a nice, single boundary, or a big smear?
> 3. If the latter, how were the data analyzed to come to his conclusion?
>
> Hopefully he can comment on these aspects.
>
> Cheerio,
> Chad
>
>
>
> ________________________________
>
> From: Kushol Gupta <[log in to unmask]>
> To: [log in to unmask]
> Sent: Fri, May 27, 2011 9:51:04 AM
> Subject: Re: [ccp4bb] larger molecular weight shown by analytic
> ultracentrifugation
>
> Hi Jerry –
>
>
>
> By AUC, do you mean sedimentation velocity (SV)?
>
>
>
> Both gel filtration and SV are not terribly great ways to determine precise
> molecular mass, especially if the macromolecule of interest is anisotropic
> in shape. In your SV values, do you see a large f/fo, or a broad
> distribution? Can you run a sedimentation equilibrium experiment? If you run
> HYDROPRO on your prospective oligomer structure, do you arrive at
> theoretical S and Rs values that jive with your solution data?
>
>
>
> A nice crosscheck you could do with the data in hand (if your measurements
> from both approaches were performed in the same buffer at the same
> temperature) is calculate the mass using the Siegel and Monty equation
> (Siegel, L. M., and Monty, K. J. (1966) Biochim. Biophys. Acta 112,
> 346–362), where the mass of the particle is calculated from Rs (from gel
> filtration) and the S(t,b) value from sedimentation velocity.
>
>
>
> Hope this helps,
>
>
>
> Kushol
>
>
>
> Kushol Gupta, Ph.D.
>
> Research Associate
>
> Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine
>
> [log in to unmask]
>
> 215-573-7260 / 267-259-0082
>
>
>
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Jerry
> McCully
> Sent: Friday, May 27, 2011 10:17 AM
> To: [log in to unmask]
> Subject: [ccp4bb] larger molecular weight shown by analytic
> ultracentrifugation
>
>
>
> Dear ALL;
>
> I am sorry for this off-topic question about analytic
> ultracentrifugation (AUC).
>
> We recently solved one structure from crystals grown out of PEG4000 plus
> buffer. Since the crystal was grown from PEG, we think the protein would
> maintain its native oligomerization state as in the solution.
>
> Indeed, the crystal packing clearly shows a tetramer of this protein.
> However, both the gel-filtration and AUC showed larger molecular weight,
> roughly around 6-mer or 7-mer.
>
> IN the crystal lattice, we could not find any 6-mer or 7-mer state.
>
> Could anyone give some comments on this discrepancy?
>
> Thanks a lot and have a nice weekend!
>
> Jerry McCully
--
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: [log in to unmask]
*******************************************
|