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
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
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
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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
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 <->
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
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
?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
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
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
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
Thank you, Eleanor, for an important reminder :
obviously, one more recent and relevant paper is that by Read and McCoy (Acta
Cryst, D, 2016)
[ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784668/ |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784668/ ]
With best wishes,
Sacha
-
The maths for estimating the FOM during refinement in REFMAC is
given in some detail in the original paper.
However the assessment uses estimates of the observation standard
uncertainly, and SigmaA - the estimate of the resolution dependent
error due to coordinate errors and missing atoms -
and
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Dear Andre,
I would strongly advice you to look at the article by Lunin and Skovoroda (Acta
Cryst, A, 1995) that addresses exactly your question:
[ https://scripts.iucr.org/cgi-bin/paper?vs0124 |
https://scripts.iucr.org/cgi-bin/paper?vs0124 ]
The authors remind a very important point that
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