[RASMB] Re: RASMB Digest, Vol 32, Issue 14
Eric Salgado
esalgado at popmail.ucsd.edu
Thu May 29 15:24:36 PDT 2008
All of the responses I have gotten to my questions this morning have
been very helpful in making me think about my problem a little more. To
address some of the points you have all brought up:
My system is not biologically relevant and my oligomeric
states (including the aggregates I described previously which are much
larger than my trimer and only about 5% of the species I see in SV) are
only induced by the addition of metal ions. Although I would ultimately
like to obtain some kind of apparent thermodynamic data from my system,
it really is more important that I can show that the trimer I see in the
crystal is also present in solution, and was hoping that the combination
of SE and SV data would stop the ambiguity that is troubling me.
I have found with other samples that if I increase my protein
concentration much more than 1 mM, my molecular mass will continue
shifting towards the aggregate. With the 1 mM data I have now, the
trimer is the main species, but this shifting trend appears to be
starting in this sample, and so I fear that increasing my concentration
may not help clear anything up.
Although I could use a self association medel in SEDPHAT, would
it instead be viable to globally fit multiple SV datasets using a
species analysis model if I hold the monomeric MW and s value constant?
The monomer s value I have obtained experimentally from an SV of the
protein with EDTA, and so no oligomers form, leaving me confident in
this value.
Using my crystal structure, how can I calculate a theoretical s
value for my trimer? If I use the calculator in sedfit, it generally
overestimates the s value of the monomeric protein which I have
observed experimentally. This isn't surprising since the protein is
rod-shaped and not spherical, but it makes interpreting the predicted s
values from sedfit rather difficult.
I think that hits upon all of the main points you have all
brought up, and sums up all the new questions I have.
Thank you again for all of your time and patience. I really appreciate it,
Eric
rasmb-request at rasmb.bbri.org wrote:
> Send RASMB mailing list submissions to
> rasmb at rasmb.bbri.org
>
> To subscribe or unsubscribe via the World Wide Web, visit
> http://rasmb.bbri.org/mailman/listinfo/rasmb
> or, via email, send a message with subject or body 'help' to
> rasmb-request at rasmb.bbri.org
>
> You can reach the person managing the list at
> rasmb-owner at rasmb.bbri.org
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of RASMB digest..."
>
>
> Today's Topics:
>
> 1. Interpreting SV and SE data (Eric Salgado)
> 2. Re: Interpreting SV and SE data (Walter Stafford)
> 3. AW: [RASMB] Interpreting SV and SE data (Titus M. Franzmann)
> 4. Re: AW: [RASMB] Interpreting SV and SE data (Eric Salgado)
> 5. RE: Interpreting SV and SE data (John Philo)
> 6. Re: Interpreting SV and SE data (Peter Schuck)
> 7. AW: AW: [RASMB] Interpreting SV and SE data (Titus M. Franzmann)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 29 May 2008 10:54:42 -0700
> From: Eric Salgado <esalgado at popmail.ucsd.edu>
> Subject: [RASMB] Interpreting SV and SE data
> To: rasmb at server1.bbri.org
> Message-ID: <483EEDE2.3050003 at popmail.ucsd.edu>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> 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
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 29 May 2008 14:43:10 -0400
> From: Walter Stafford <stafford at bbri.org>
> Subject: Re: [RASMB] Interpreting SV and SE data
> To: Eric Salgado <esalgado at popmail.ucsd.edu>
> Cc: rasmb at server1.bbri.org
> Message-ID: <483EF93E.3050103 at bbri.org>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
>
> Hi Eric,
>
> I don't think c(MW) is an appropriate way to analyze an interacting
> system. In the case of a self-associating system the boundary is called
> a reaction boundary and is not composed in individual peaks that can be
> resolved by any method. Molecular weight values obtained from c(MW)
> would not have any meaning in that case. So I would be very suspicious
> of a trimer molecular weight value obtained that way.
>
> The boundary must be treated using one of the methods for whole
> boundary fitting to solutions of the Lamm equation for an interacting
> system. And the fit must be done globally over the widest range of
> concentrations possible to get reliable estimates of the stoichiometry
> and equilibrium constants. You could use either SEDPHAT or SEDANAL for
> global fitting to the various models you want to try. SEDANAL has a
> Model Editor that allows you to specify a very wide variety of arbitrary
> association schemes.
>
> SE is a good way to verify the SV results but in your case it sounds
> like you need to go to higher concentrations to populate the larger
> species in order to resolve the ambiguity between monomer-dimer and
> monomer -dimer-trimer. It is well known also that a
> monomer-dimer-tetramer system can masquerade as a monomer-trimer system
> if the concentration range is not high enough. Solubility issues may
> prevent you from increasing the concentration enough, though.
>
>
> Walter Stafford
>
> Eric Salgado wrote:
>
>> 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
>>
>>
>
>
More information about the RASMB
mailing list