[RASMB] Help needed : follow up

Ewa Folta-Stogniew ef55 at email.med.yale.edu
Thu Aug 30 08:13:43 PDT 2012


Dear Indrani,

if you were at 500 nM concentration , you were at ~10xKd so you 
should see only dimer.  You need to go lower with concentration to 
see the dissociation process.

For 30kDa protein, you should be able to go to 10 ug/ml at the apex, 
which is 0.3nM if my math is correct (1-2 ug/ml if your system is 
very well maintained, which will be 0.03nM), thus, for 60 nM Kd you 
should see change in Mw across the peak.  If as Tom mentioned you 
have irreversible dimer present, you should see that as well- 
especially is this is a time-dependent phenomena your SEC/MALLS 
results should be in accord with your AUC data.

If you can afford to send some protein here, I can have a look at 
it.  We had seen 25 kDa protein dimerizing with Kd of 25nM, so the 
system was comparable to yours, and I had not even needed FL for 
that, but I have one in case I need to monitor protein below UV 
detection limit.

Ewa

At 10:59 AM 8/30/2012, Sen Indrani wrote:
>Dear Ewa,
>      Thank you for your comments. The lowest I went down with SEC 
> MALS is 0.5mg/ml(16uM) with a good signal to noise ratio and at 
> 0.5uM at the peak maxima. I couldnt go below the Kd as the noise 
> was too high than for a good mass determination. The monomer is 
> 30kDa. We do have a HPLC-FL  but I am not sure about the signal to 
> noise limitation of this instrument as well. I would surely check 
> that out. Unfortunately we do not have FL detection on our SEC-MALS.
>Regards
>Indrani
>
>
>
>----------
>From: Ewa Folta-Stogniew [mailto:ef55 at email.med.yale.edu]
>Sent: 30 August 2012 15:02
>To: Sen Indrani; rasmb at rasmb.bbri.org
>Subject: [RASMB] Help needed : follow up
>
>Dear Indrani,
>
>SEC/MALLS with FL detection should complement your AUC with FL 
>detection in terms of time-dependent changes (if there are any) in 
>dimer stability.  It would be rather unlikely that the dimer falls 
>apart into partially unfolded monomers that have the same 
>hydrodynamic size as a dimer.
>
>On a well maintained SEC/MALLS system you can go down to 1ug/ml 
>concentration at teh apex of the eluting peak to measure the Mw from 
>SEC/MALLS experiment (and Kd of 60nM can be measured by SEC/MALLS 
>unless you have very unusual kinetics, but if your Kd changes with 
>time, it should be picked up by SEC/MALLS as well).  In addition, 
>you can follow dimer dissociation to much smaller concentrations 
>using FL detection to monitor peak elution position; from the shift 
>in the peak elution position you can compute dissociation constant. 
>I do not know it for a fact, but I would guess that  FL detection on 
>HPLC FL detector can probably go to lower concentrations of protein 
>than FL detection on the AUC system, or at minimum match the 
>sensitivity you have on AUC.    You simply need to assure that the 
>injection volume to are constant, so all the shift in elution is due 
>to size changes.
>
>how low had you gotten with protein concentrations during your 
>SEC/MALLS experiments?   What are the concentrations in your eluting 
>peak at the apex of that peak in mg/ml and uM? Have you gotten below 
>you Kd value for your SEC/MALLS?  How big is your monomer?
>
>Ewa
>
>At 05:13 AM 8/30/2012, Sen Indrani wrote:
>
>>  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
>>
>>-----Original Message-----
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>>On Behalf Of rasmb-request at rasmb.bbri.org
>>Sent: 24 August 2012 18:00
>>To: rasmb at rasmb.bbri.org
>>Subject: RASMB Digest, Vol 35, Issue 4
>>
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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)
>>
>>
>>----------------------------------------------------------------------
>>
>>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>
>>Content-Type: text/plain; charset="us-ascii"
>>
>>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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>>------------------------------
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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
>>Message-ID: <7.0.1.0.2.20120823161225.07f6e2a8 at email.med.yale.edu>
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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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