Re: [ccp4bb] ITC question -dimer vs monomer

[Bonsor, Daniel]

You may want to look at the following paper concerning Il8, dimerization, 
binding and ITC.

http://m.jbc.org/content/279/35/36175.full

Get Outlook for Android<https://aka.ms/ghei36>


From: CCP4 bulletin board  on behalf of Bernhard Rupp 

Sent: Friday, October 4, 2019 5:06:10 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Thanks to All for the extended & informative responses. If true thermodynamic 
equilibria
are realized, then I would agree that regardless of the pathway the endpoint 
(or integrated H)
should be the same.  The actual pathway and cooperativity probably will make 
this an
interesting problem. I may keep bugging selected victims off-board once I have 
the first data.

Many thanks again, BR

From: CCP4 bulletin board  On Behalf Of Barone, Matthias
Sent: Thursday, October 3, 2019 18:59
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer


As Reza already pointed out, ITC cannot tell you anything about the 
sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> 
AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a 
fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2.

Given the lack of additional information, its probably good to assume a simple 
one-to-one model and titrate either partner to the other.

If you have two separate binding steps (with similar Kd for B2 for A as well as 
AB2 for A), you would measure an apparent affinity and would see the 
stochiometrics according to  the inflection point (be it around equimolar 
excess or at 0.5 or 2, depending on whether you titrate A or B). If the 
reaction is more complicated and the the affinities for B2 for A differ 
significantly much from the affinity of AB2 for A, then a simple one-to-one 
would leave some notable information in the residual standard deviations 
(meaning, the residuals would not spread normally around the Regression line, 
but should show a wavy pattern).

Sorry for the long mail..

Matthias



Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284
future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$>
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] ITC question -dimer vs monomer

[Bernhard Rupp]

Thanks to All for the extended & informative responses. If true
thermodynamic equilibria

are realized, then I would agree that regardless of the pathway the endpoint
(or integrated H)

should be the same.  The actual pathway and cooperativity probably will make
this an 

interesting problem. I may keep bugging selected victims off-board once I
have the first data.

 

Many thanks again, BR

 

From: CCP4 bulletin board  On Behalf Of Barone,
Matthias
Sent: Thursday, October 3, 2019 18:59
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

 

As Reza already pointed out, ITC cannot tell you anything about the
sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <->
AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to
a fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2. 

Given the lack of additional information, its probably good to assume a
simple one-to-one model and titrate either partner to the other.

If you have two separate binding steps (with similar Kd for B2 for A as well
as AB2 for A), you would measure an apparent affinity and would see the
stochiometrics according to  the inflection point (be it around equimolar
excess or at 0.5 or 2, depending on whether you titrate A or B). If the
reaction is more complicated and the the affinities for B2 for A differ
significantly much from the affinity of AB2 for A, then a simple one-to-one
would leave some notable information in the residual standard deviations
(meaning, the residuals would not spread normally around the Regression
line, but should show a wavy pattern). 

Sorry for the long mail..

Matthias

 

Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284

future.

 

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK> > On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> 
Subject: [ccp4bb] ITC question -dimer vs monomer

 

Hi Fellows,

 

please let me ask the respective experts an ITC question: I have 2 proteins,
stable and dialyzed in identical buffer.

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
dimer will form.

 

Intuitively, it should make a difference whether I titrate the dimer with
the monomer or vice versa.

In the first case, a momomer would initially meet a lot of free dimers, and
I would expect that randomly, a AB2 complex

is more likely to form than a A2B2 (let’s disregard any more complex
colligative/cooperative effects).

 

If I drip the dimer into the monomer pool, it is quite likely that the B
dimer meets 2 free As, and I get right away a higher population of A2B2s.

 

Maybe at dilutions of ITC and with sufficient equilibration that is not an
issue at all (again, absent any cooperative effects that might alter the
first Kd vs. the second, despite the sites on the dimer are at least
initially equivalent).

 

Can someone guide me towards literature about this or perhaps share some
first-hand experience?

 

Many thanks, BR

 

--

Bernhard Rupp

http://www.hofkristallamt.org/
<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE
9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$> 

b...@hofkristallamt.org <mailto:b...@hofkristallamt.org> 

+1 925 209 7429

+43 676 571 0536

--

Many plausible ideas vanish 

at the presence of thought

--

 

 

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Re: [ccp4bb] ITC question -dimer vs monomer

[DUMAS Philippe (IGBMC)]

Dear Bernhard 
I share your intuition: we should expect to observe different shapes of 
titration curves depending on whether A or B2 is in the syringe (titration and 
reverse titration). 
I suppose that you want to test your hypothesis that, eventually, you get A2B2. 
Independently of any kinetic considerations , one should consider the 
possibility that the successive equilibria A+B2<-->AB2 and A + AB2 <-->A2B2 
have distinct Kds and distinct DeltaH (which would of course be more favorable 
to detect these two distinct binding events, if they exist). 
I therefore suggest that you perform both possible titrations (if each protein 
can be sufficiently concentrated to be in the syringe) and that you try to fit 
each titration curve with the same model of interaction. If this works well, 
then the must would be to fit both titration curves at the same time with the 
same model of interaction . This is certainly the most demanding method to test 
a hypothesis and this is not at all equivalent to fit independently the two 
kinds of titration curves as you can imagine. (Let's say that this would amount 
to refine a single molecular model against crystal data from two space 
groups).The problem is of the practical possibility of doing such a joint fit 
with the available programs. I personally do such things with my own (not 
user-friendly!) programs. As far a I know, this is not possible, neither with 
Origin or PEAQ from Malvern, nor with NanoAnalyze from TA. I know that 
AFFINImeter is quite flexible to allow using specific models, but I'm not sure 
it would allow you to make such a global fit. I don't know about the 
possibility of SEDPHAT developed by P. Schuck. 
I hope this fits with your expectations. 
Best 
Philippe Dumas 



De: "Bernhard Rupp"  
À: CCP4BB@JISCMAIL.AC.UK 
Envoyé: Jeudi 3 Octobre 2019 17:05:56 
Objet: [ccp4bb] ITC question -dimer vs monomer 



Hi Fellows, 



please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer. 

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form. 



Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa. 

In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex 

is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects). 



If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s. 



Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent). 



Can someone guide me towards literature about this or perhaps share some 
first-hand experience? 



Many thanks, BR 



-- 

Bernhard Rupp 

[ http://www.hofkristallamt.org/ | http://www.hofkristallamt.org/ ] 

[ mailto:b...@hofkristallamt.org | b...@hofkristallamt.org ] 

+1 925 209 7429 

+43 676 571 0536 

-- 

Many plausible ideas vanish 

at the presence of thought 

-- 






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Re: [ccp4bb] ITC question -dimer vs monomer

[Barone, Matthias]

No, the law of mass action does not depend on whether A is titrated into B or 
vice versa. The heat measured is proportional to the complex formed, relative 
to one partner being kept at constant concentration (the referece in the cell). 
The law of mass action can be re-written to describe the complex concentration 
itself as a function of A, B and Kd. Assuming a one-to-one stoichiometry, the 
function contains sums of each variable being substracted by a root function:

AB = 0.5 [ Atotal + Btotal + Kd - sqrt{  (Atotal + Btotal + Kd)^2 - 
4*Atotal*Btotal   }]

It is correct that these values should be roughly in the same range. This, 
however, is not restricted to ITC solely, but to HSQC or fluorescence-based 
titrations too. Its a direct consequence of the law of mass action.

In order to fully saturate either binding partner, say fully saturate A; AB/A 
approaching 1, all three factors need to be in the same range. The same would 
hold with B in the cell, saturating AB/B. The underlaying mass action is the 
same, just the non-ligated reference is exchanged.

For a stochiometry different from 1:1, the mass action does not yield in an 
explicit solution like the one written above. To force such a solution, one 
concentration is multiplied by a factor, N, assuming N independent, 
non-cooperative binding sites for N ligands. Lets assume a 1:2 in your case, 
2A+B2 <-> A2B2. Substituting Btotal with N*Btotal would allow to assume a 
one-to-one model with N=2. If you decide to exchange the titrant, N now 
corrects the other partner, yielding in N=0.5.





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Roger Rowlett 

Sent: Thursday, October 3, 2019 8:36:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Won't this depend on the relative final concentrations of A and B in the two 
experiments? If A going into excess B will have different mass action 
considerations that B going into excess A. Even if the final concentrations of 
A and B are stoichiometric, the initial stages of the titration will have very 
different mass action products for A into B vs. B into A. An additional wrinkle 
is the concentrations of A and B relative to the dissociation constant Kd. The 
titration curve math gets a little more complex when the concentration of the 
species is in the same order of magnitude as the Kd. There are quite a few 
examples of bollixed binding curves in the literature for tight-binding 
equilibria that ignore the relationship between Kd and ligand concentrations.  
Cooperativity issues will of course perturb any pure, non-cooperative 
statistical analysis based on equilibrium constants.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu<mailto:rrowl...@colgate.edu>

On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp 
mailto:hofkristall...@gmail.com>> wrote:
I am not looking for anything yet – I wonder what – if any – the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don’t understand what you are trying to do—are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively,

Re: [ccp4bb] ITC question -dimer vs monomer

[Roger Rowlett]

Won't this depend on the relative final concentrations of A and B in the
two experiments? If A going into excess B will have different mass action
considerations that B going into excess A. Even if the final concentrations
of A and B are stoichiometric, the initial stages of the titration will
have very different mass action products for A into B vs. B into A. An
additional wrinkle is the concentrations of A and B relative to the
dissociation constant Kd. The titration curve math gets a little more
complex when the concentration of the species is in the same order of
magnitude as the Kd. There are quite a few examples of bollixed binding
curves in the literature for tight-binding equilibria that ignore the
relationship between Kd and ligand concentrations.  Cooperativity issues
will of course perturb any pure, non-cooperative statistical analysis based
on equilibrium constants.

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
email: rrowl...@colgate.edu

On Thu, Oct 3, 2019 at 11:51 AM Bernhard Rupp 
wrote:

> I am not looking for anything yet – I wonder what – if any – the
> consequences of doing it one way or the other would be.
>
> I am reasonably certain that any difference affects the analysis.
>
>
>
> Thx, BR
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Keller,
> Jacob
> *Sent:* Thursday, October 3, 2019 17:41
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] ITC question -dimer vs monomer
>
>
>
> I don’t understand what you are trying to do—are you trying to show, by
> the difference in ITC response, that the predictions you made about the
> oligomerization are true?
>
>
>
> JPK
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> Rupp
> *Sent:* Thursday, October 3, 2019 11:06 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] ITC question -dimer vs monomer
>
>
>
> Hi Fellows,
>
>
>
> please let me ask the respective experts an ITC question: I have 2
> proteins, stable and dialyzed in identical buffer.
>
> A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
> dimer will form.
>
>
>
> Intuitively, it should make a difference whether I titrate the dimer with
> the monomer or vice versa.
>
> In the first case, a momomer would initially meet a lot of free dimers,
> and I would expect that randomly, a AB2 complex
>
> is more likely to form than a A2B2 (let’s disregard any more complex
> colligative/cooperative effects).
>
>
>
> If I drip the dimer into the monomer pool, it is quite likely that the B
> dimer meets 2 free As, and I get right away a higher population of A2B2s.
>
>
>
> Maybe at dilutions of ITC and with sufficient equilibration that is not an
> issue at all (again, absent any cooperative effects that might alter the
> first Kd vs. the second, despite the sites on the dimer are at least
> initially equivalent).
>
>
>
> Can someone guide me towards literature about this or perhaps share some
> first-hand experience?
>
>
>
> Many thanks, BR
>
>
>
> --
>
> Bernhard Rupp
>
> http://www.hofkristallamt.org/
> <https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$>
>
> b...@hofkristallamt.org
>
> +1 925 209 7429
>
> +43 676 571 0536
>
> --
>
> Many plausible ideas vanish
>
> at the presence of thought
>
> --
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> <https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1

Re: [ccp4bb] ITC question -dimer vs monomer

[Rajiv gandhi.s]

Dear Dr.BR

There could be two possibilities,  you could try to do titration of dimer
into monomer loaded in cell or vice versa, and with subtraction of heat of
dilution from protein-protein titration. These kind of molecular
interaction involving distinct oligomer units need be addressed  by any
other orthogonal methods.

Regards
Rajivgandhi Sundaram


On Thu, Oct 3, 2019, 10:21 PM clare stevenson (JIC) <
clare.steven...@jic.ac.uk> wrote:

> I would try it both ways and see what you get.  Also do controls of buffer
> into each protein
>
>
>
> For extra info could also try with SPR.  Always best to do these things
> using multiple complimentary methods
>
>
>
> Best wishes
>
>
>
> Clare
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Bernhard
> Rupp
> *Sent:* 03 October 2019 16:06
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] ITC question -dimer vs monomer
>
>
>
> Hi Fellows,
>
>
>
> please let me ask the respective experts an ITC question: I have 2
> proteins, stable and dialyzed in identical buffer.
>
> A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
> dimer will form.
>
>
>
> Intuitively, it should make a difference whether I titrate the dimer with
> the monomer or vice versa.
>
> In the first case, a momomer would initially meet a lot of free dimers,
> and I would expect that randomly, a AB2 complex
>
> is more likely to form than a A2B2 (let’s disregard any more complex
> colligative/cooperative effects).
>
>
>
> If I drip the dimer into the monomer pool, it is quite likely that the B
> dimer meets 2 free As, and I get right away a higher population of A2B2s.
>
>
>
> Maybe at dilutions of ITC and with sufficient equilibration that is not an
> issue at all (again, absent any cooperative effects that might alter the
> first Kd vs. the second, despite the sites on the dimer are at least
> initially equivalent).
>
>
>
> Can someone guide me towards literature about this or perhaps share some
> first-hand experience?
>
>
>
> Many thanks, BR
>
>
>
> --
>
> Bernhard Rupp
>
> http://www.hofkristallamt.org/
>
> b...@hofkristallamt.org
>
> +1 925 209 7429
>
> +43 676 571 0536
>
> --
>
> Many plausible ideas vanish
>
> at the presence of thought
>
> --
>
>
>
>
> --
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Re: [ccp4bb] ITC question -dimer vs monomer

[Barone, Matthias]

As Reza already pointed out, ITC cannot tell you anything about the 
sub-processes that underlay an equilibrium, be it complicated like 2A+B2 <-> 
AB2 + A <-> A2B2, or with (virtually) no intermediate 2A+B2 <-> A2B2 due to a 
fast second forward reaction , or a simple one-to-one model A+B2 <-> AB2.

Given the lack of additional information, its probably good to assume a simple 
one-to-one model and titrate either partner to the other.

If you have two separate binding steps (with similar Kd for B2 for A as well as 
AB2 for A), you would measure an apparent affinity and would see the 
stochiometrics according to  the inflection point (be it around equimolar 
excess or at 0.5 or 2, depending on whether you titrate A or B). If the 
reaction is more complicated and the the affinities for B2 for A differ 
significantly much from the affinity of AB2 for A, then a simple one-to-one 
would leave some notable information in the residual standard deviations 
(meaning, the residuals would not spread normally around the Regression line, 
but should show a wavy pattern).

Sorry for the long mail..

Matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: CCP4 bulletin board  on behalf of Michael Fairhead 

Sent: Thursday, October 3, 2019 5:59:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

Hello,
If you Google Alan cooper ITC insulin, you should find his work describing the 
study of insulin dimer:monomer equilibrium and the effect of cyclodextrin 
studied via ITC. This may be of some help.
Cheers
Mike

https://www.google.com/url?sa=t=web=j=http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf=2ahUKEwiOpoeisYDlAhUDThUIHX5mAyMQFjAAegQIAhAB=AOvVaw0Ng-W03upy4DG12unFGDY3


From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet – I wonder what – if any – the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don’t understand what you are trying to do—are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let’s disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$>
b...@hofkristallamt.org<mailto:

Re: [ccp4bb] ITC question -dimer vs monomer

[clare stevenson (JIC)]

I would try it both ways and see what you get.  Also do controls of buffer into 
each protein

For extra info could also try with SPR.  Always best to do these things using 
multiple complimentary methods

Best wishes

Clare

From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:06
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] ITC question -dimer vs monomer

[Michael Fairhead]

Hello,
If you Google Alan cooper ITC insulin, you should find his work describing the 
study of insulin dimer:monomer equilibrium and the effect of cyclodextrin 
studied via ITC. This may be of some help.
Cheers
Mike

https://www.google.com/url?sa=t=web=j=http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf=2ahUKEwiOpoeisYDlAhUDThUIHX5mAyMQFjAAegQIAhAB=AOvVaw0Ng-W03upy4DG12unFGDY3


From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: 03 October 2019 16:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet - I wonder what - if any - the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$>
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1<https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStkIPgsQE$>



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] ITC question -dimer vs monomer

[Reza Khayat]

?Isn't the entire idea of using ITC that you are measuring an equilibrium 
constant? Wouldn't this eliminate the nuances of what you're looking for (i.e. 
kinetics)? Perhaps you should use SPR to tease out this model?


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031

From: CCP4 bulletin board  on behalf of Bernhard Rupp 

Sent: Thursday, October 3, 2019 11:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] Re: [ccp4bb] ITC question -dimer vs monomer

I am not looking for anything yet - I wonder what - if any - the consequences 
of doing it one way or the other would be.
I am reasonably certain that any difference affects the analysis.

Thx, BR

From: CCP4 bulletin board  On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.proofpoint.com/v2/url?u=https-3A__urldefense.com_v3_-5F-5Fhttp-3A_www.hofkristallamt.org_-5F-5F-3B-21oCotSwSxbw8-21SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs-24=DwMFAg=4NmamNZG3KTnUCoC6InoLJ6KV1tbVKrkZXHRwtIMGmo=1DzJFW0v6TgEhkW1gy_-ke-RbtvS1fzEbD5_hcb9Up0=7ZuSeTWtLa-zqSUcsmMCVR2swEqxCFprx15uq21CKHk=Lxm2SJmbFVHs4eyFZrLgqJ3_PkrVzZDYhsWUJPWKpdE=>
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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Re: [ccp4bb] ITC question -dimer vs monomer

[Bernhard Rupp]

I am not looking for anything yet - I wonder what - if any - the
consequences of doing it one way or the other would be.

I am reasonably certain that any difference affects the analysis.

 

Thx, BR

 

From: CCP4 bulletin board  On Behalf Of Keller, Jacob
Sent: Thursday, October 3, 2019 17:41
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] ITC question -dimer vs monomer

 

I don't understand what you are trying to do-are you trying to show, by the
difference in ITC response, that the predictions you made about the
oligomerization are true?

 

JPK

 

+

Jacob Pearson Keller

Research Scientist / Looger Lab

HHMI Janelia Research Campus

19700 Helix Dr, Ashburn, VA 20147

Desk: (571)209-4000 x3159

Cell: (301)592-7004

+

 

The content of this email is confidential and intended for the recipient
specified in message only. It is strictly forbidden to share any part of
this message with any third party, without a written consent of the sender.
If you received this message by mistake, please reply to this message and
follow with its deletion, so that we can ensure such a mistake does not
occur in the future.

 

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK> > On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK> 
Subject: [ccp4bb] ITC question -dimer vs monomer

 

Hi Fellows,

 

please let me ask the respective experts an ITC question: I have 2 proteins,
stable and dialyzed in identical buffer.

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
dimer will form.

 

Intuitively, it should make a difference whether I titrate the dimer with
the monomer or vice versa.

In the first case, a momomer would initially meet a lot of free dimers, and
I would expect that randomly, a AB2 complex

is more likely to form than a A2B2 (let's disregard any more complex
colligative/cooperative effects).

 

If I drip the dimer into the monomer pool, it is quite likely that the B
dimer meets 2 free As, and I get right away a higher population of A2B2s.

 

Maybe at dilutions of ITC and with sufficient equilibration that is not an
issue at all (again, absent any cooperative effects that might alter the
first Kd vs. the second, despite the sites on the dimer are at least
initially equivalent).

 

Can someone guide me towards literature about this or perhaps share some
first-hand experience?

 

Many thanks, BR

 

--

Bernhard Rupp

http://www.hofkristallamt.org/
<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE
9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$> 

b...@hofkristallamt.org <mailto:b...@hofkristallamt.org> 

+1 925 209 7429

+43 676 571 0536

--

Many plausible ideas vanish 

at the presence of thought

--

 

 

  _  

To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] ITC question -dimer vs monomer

[Keller, Jacob]

I don't understand what you are trying to do-are you trying to show, by the 
difference in ITC response, that the predictions you made about the 
oligomerization are true?

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
specified in message only. It is strictly forbidden to share any part of this 
message with any third party, without a written consent of the sender. If you 
received this message by mistake, please reply to this message and follow with 
its deletion, so that we can ensure such a mistake does not occur in the future.

From: CCP4 bulletin board  On Behalf Of Bernhard Rupp
Sent: Thursday, October 3, 2019 11:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ITC question -dimer vs monomer

Hi Fellows,

please let me ask the respective experts an ITC question: I have 2 proteins, 
stable and dialyzed in identical buffer.
A is a monomer and B an obligate dimer. I suspect that eventually a A2B2 dimer 
will form.

Intuitively, it should make a difference whether I titrate the dimer with the 
monomer or vice versa.
In the first case, a momomer would initially meet a lot of free dimers, and I 
would expect that randomly, a AB2 complex
is more likely to form than a A2B2 (let's disregard any more complex 
colligative/cooperative effects).

If I drip the dimer into the monomer pool, it is quite likely that the B dimer 
meets 2 free As, and I get right away a higher population of A2B2s.

Maybe at dilutions of ITC and with sufficient equilibration that is not an 
issue at all (again, absent any cooperative effects that might alter the first 
Kd vs. the second, despite the sites on the dimer are at least initially 
equivalent).

Can someone guide me towards literature about this or perhaps share some 
first-hand experience?

Many thanks, BR

--
Bernhard Rupp
http://www.hofkristallamt.org/<https://urldefense.com/v3/__http:/www.hofkristallamt.org/__;!oCotSwSxbw8!SE9mT6grUy1tHFSKLCraXt4bhlDri03OEMEyqQUCLAVSLsg3vwn0GTQtxbStgtNvxBs$>
b...@hofkristallamt.org<mailto:b...@hofkristallamt.org>
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--




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[ccp4bb] ITC question -dimer vs monomer

[Bernhard Rupp]

Hi Fellows,

 

please let me ask the respective experts an ITC question: I have 2 proteins,
stable and dialyzed in identical buffer.

A is a monomer and B an obligate dimer. I suspect that eventually a A2B2
dimer will form.

 

Intuitively, it should make a difference whether I titrate the dimer with
the monomer or vice versa.

In the first case, a momomer would initially meet a lot of free dimers, and
I would expect that randomly, a AB2 complex

is more likely to form than a A2B2 (let's disregard any more complex
colligative/cooperative effects).

 

If I drip the dimer into the monomer pool, it is quite likely that the B
dimer meets 2 free As, and I get right away a higher population of A2B2s.

 

Maybe at dilutions of ITC and with sufficient equilibration that is not an
issue at all (again, absent any cooperative effects that might alter the
first Kd vs. the second, despite the sites on the dimer are at least
initially equivalent).

 

Can someone guide me towards literature about this or perhaps share some
first-hand experience?

 

Many thanks, BR

 

--

Bernhard Rupp

http://www.hofkristallamt.org/

b...@hofkristallamt.org  

+1 925 209 7429

+43 676 571 0536

--

Many plausible ideas vanish 

at the presence of thought

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