[RASMB] Help needed : follow up

Thu Aug 30 02:13:57 PDT 2012

 Dear All,
               Thank you Tom, Sophia and Ewa for the replies. I already
had done the SEC_MALS experiment and the mass distribution under the
peak was always homogeneous. The protein is a dimer always as it has a
coiled coil, but the dimerization is concentration dependent. I also did
CD of the protein and the melting curve is completing reversible . The
SEC peak is also almost symmetric with no major trailing or leading
edge. I am limited by the SEC MALS with going down with the
concentration so I have to use FDS_SV in AUC only . The Kd would
defintely be in the nanomolar range and so the question I asked you
before still remains a question. Please could you provide further
insights.

Thank you,
Regards
Indrani

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Today's Topics:

   1. Re: Help needed (Sophia Kenrick)
   2. Re: Help needed (Ewa Folta-Stogniew)
   3. Re: Help needed (Borries Demeler)

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Message: 1
Date: Thu, 23 Aug 2012 12:44:56 -0700
From: Sophia Kenrick <skenrick at wyatt.com>
To: "rasmb at rasmb.bbri.org" <rasmb at rasmb.bbri.org>
Subject: Re: [RASMB] Help needed
Message-ID:

<0E627C11B80B30409FFE2FBB526943ED022D90F369FA at exchange2007.wyatt.com>
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Dear Indrani,

As Tom mentioned, definitely look into the possibility of irreversible
aggregation happening in parallel with your reversibly associating
protein.  SEC combined with multi-angle light scattering is an ideal
method for identifying the molecular weight of each species eluted from
the column.  In the case of irreversible association, the aggregate peak
should be completely separate from the monomer peak.  You can perform an
SEC-MALS run on an aliquot of your protein after incubating for 1-15
hours to see if the fraction of irreversible aggregate grows with time.

In the case of reversible association, you should see an increase in Mw
across a single peak as compared to the expected monomer molar mass for
your protein.  For a reversibly associating protein, the main peak in an
SEC chromatogram will shift to higher Mw as you inject a higher
concentration on the column (or larger volume of the same
concentration).  Depending on the kinetics of the reaction, you may be
able to measure a higher molar mass at the very apex of the peak (where
the concentration is highest) as compared to the leading or trailing
edge where the concentration is lower.  However, often you get a
constant, average Mw across the peak that changes as a function of the
amount of protein that was loaded on the column.  You can get a
semi-quantitative estimate of the KD from this type of experiment.

A more accurate quantification of oligomerization KD by light scattering
would be performed in batch.  Composition gradient static light
scattering (CG-SLS or CG-MALS) involves measuring the light scattering
signal of different concentrations of an unfractionated macromolecular
solution.  As complexes form, the weight average molar mass of the
solution should increase.  The change in light scattering (Mw) as a
function of composition can then be fit to an appropriate association
model to yield affinity and stoichiometry.  Below is a link to a book
chapter reviewing this technique, and the references listed in it may be
useful to you.

http://www.intechopen.com/books/protein-interactions/characterization-of
-protein-protein-interactions-via-static-and-dynamic-light-scattering

Best regards,
Sophia

Sophia Kenrick, Ph.D. | Application Development Engineer Wyatt
Technology Corporation 6300 Hollister Avenue | Santa Barbara, CA
93117-3253
Phone:  (805) 681-9009 x297 | Fax:  (805) 681-0123
Web:  www.wyatt.com<http://www.wyatt.com> | E-mail:
skenrick at wyatt.com<mailto:skenrick at wyatt.com>

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Message: 2
Date: Thu, 23 Aug 2012 16:21:49 -0400
From: Ewa Folta-Stogniew <ef55 at email.med.yale.edu>
To: Sophia Kenrick <skenrick at wyatt.com>,	"rasmb at rasmb.bbri.org"
	<rasmb at rasmb.bbri.org>,	Sen Indrani <Indrani.Sen at psi.ch>
Subject: Re: [RASMB] Help needed
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Message: 3
Date: Thu, 23 Aug 2012 17:56:49 -0500
From: Borries Demeler <demeler at biochem.uthscsa.edu>
To: Ewa Folta-Stogniew <ef55 at email.med.yale.edu>
Cc: RASMB List <rasmb at server1.bbri.org>
Subject: Re: [RASMB] Help needed
Message-ID: <20120823225649.GA17138 at biochem.uthscsa.edu>
Content-Type: text/plain; charset=us-ascii

On Thu, Aug 23, 2012 at 04:21:49PM -0400, Ewa Folta-Stogniew wrote:

> _______________________________________________
> At 03:44 PM 8/23/2012, Sophia Kenrick wrote:
>
>   Depending on the kinetics of the reaction, you may be able to
measure a
>   higher molar mass at the very apex of the peak (where the
concentration is
>   highest) as compared to the leading or trailing edge where the
>   concentration is lower.  However, often you get a constant, average
Mw
>   across the peak that changes as a function of the amount of protein
that
>   was loaded on the column.
>
>   and:
>
>   You can get a semi-quantitative estimate of the K[D] from this type
of
>   experiment.
>
>   the last sentence is not true; depending on the kinetics of the
>   association, you can get quantitative estimate of Kd for
self-association-
>   you just need to prove that this is the case.
>
>   Wouldn't the kinetics of self-association be a concern for use of
sed.
>   velocity for Kd determination as well?
>
>   Ewa

It's not really a concern when all you want is a Kd. For purposes of
determining rate constants, the question is whether the reaction is too
fast compared to the time scale of the velocity experiment. Whether it
is or not depends on the size of the molecule and the speed with which
you are running.  If the reaction is too fast, you simply cannot
determine the rate constant of the reaction, but the Kd will still be
measurable. In the extreme case of non-interaction, you will simply get
two boundaries corresponding to monomer and oligomer.  (for kd
calculation you need molar concentrations, and you need to keep in mind
that the dimer absorbs twice as much as the monomer)

In the other extreme, when the reaction is faster than the time scale of
the vcelocity experiment, you get a reaction boundary, which changes
gradually from monomer towards oligomer as you go up in the
concentration gradient. In my experience for a typical protein the upper
limit of a k_off you can measure in a velocity experiment at 60krpm is a
rather slow speed of about 10^-4/sec. For all reactions, the Kd should
still be easily measurable as long as you have enough signal from both
the monomer and the oligomer.

-b.

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