AW: AW: [RASMB] Interpreting SV and SE data

[Titus M. Franzmann]

Eric, 
You mention that you observe a monomer-dimer equilibrium at intermediate
concentrations, which would be around 100-500 µM? And trimers only at even
higher concentrations. This indicates a very weak association constant. I
wonder, since the trimer is not populated a low concentrations (5 µM, which
typically already exceeds the cellular concentration of a certain compound),
how does it relate to a physiological relevant concentration in the cell?
Anyway, if the potential trimer is populated specifically under equilibrium
conditions, one would expect a cooperative association transition. However,
this may be hard to address with SE, as it is hard to accurately evaluate
the contribution of each species to the observed exponential, since you
propose a stable dimeric equilibrium intermediate. In SV, I guess (but this
is a question that I would like to post to the entire forum) it would
presumably give you a sigmoidal curve for interacting systems when you
analyze the observed S-values as a function of the protein concentration, as
the signal is predominantly caused by the most abundant species, which
increases relatively compared to the other species. From this you should be
able to extract the S for the species at which the association stops at a
defined level (trimer) and this should allow you to determine the KD for
each and the overall reaction. With this and the knowledge of the dimer
intermediate in this particular folding mechanism you can calculate the
fraction of trimers at any given concentration.

Best regards
Titus 


-----Ursprüngliche Nachricht-----
Von: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] Im
Auftrag von Eric Salgado
Gesendet: Donnerstag, 29. Mai 2008 21:04
Cc: rasmb at server1.bbri.org
Betreff: Re: AW: [RASMB] Interpreting SV and SE data

Titus,

    My ranges are from 5 uM to 1 mM ( viscosity at the higher end 
doesn't seem to be a problem).

Titus M. Franzmann wrote:
> Hi Eric, 
> may I ask what the concentration range is you cover in your experiments?
> Best regards
> Titus
>
> -----Ursprüngliche Nachricht-----
> Von: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] Im
> Auftrag von Eric Salgado
> Gesendet: Donnerstag, 29. Mai 2008 19:55
> An: rasmb at server1.bbri.org
> Betreff: [RASMB] Interpreting SV and SE data
>
> Hello all,
>    I have a system for which I've done SV experiments at multiple 
> concentrations that, on  increasing the concentration, reveals a peak 
> that starts at an s value of about 2, shifts to a plateau between 2.4 
> and 2.6, then steadily increases at 2.6. I should point out that all of 
> these peaks are very broad.
>    The c(MW) analysis describes a monomer at low concentrations; dimer 
> at intermediate concentrations;  something between  dimer and trimer at 
> high concentrations;  and finally, as  an aggregate  starts to form at 
> very high concentrations, a trimer peak is resolved. This trimer peak 
> correlates to our crystal structure, but it appears as if, because of 
> the apparently low association constant and fast kinetics involved with 
> the trimer formation, this fact is masked in the SV data.
>    In order to hopefully rid of this ambiguity, I performed SE 
> experiments in the concentration regime that did not form an aggregate 
> (and therefore apparently only a small amount of trimer), but find that, 
> as far as a species analysis goes, both a model with monomer-dimer or 
> monomer-dimer-trimer present are statistically equal. That being said, 
> the statistics are lower for the later model, while the fit and 
> residuals for both are equally good.
>    With all of this in hand, how can I determine the best SE model to 
> use, and can I say with certainty that the trimer exists in solution 
> from these data?
>
> I know this is a lot, but I would appreciate any help I can get  with 
> this problem.
>
> Thank you in advance,
>
> Eric Salgado
> _______________________________________________
> RASMB mailing list
> RASMB at rasmb.bbri.org
> http://rasmb.bbri.org/mailman/listinfo/rasmb
>
>   

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