simulation of exchange
Joel Mackay (j.mackay@biochem.usyd.edu.au)
Tue, 10 Aug 1999 11:34:38 +1000
Hi everyone,
We are using a 15N-HSQC titration to look at the binding interaction
between two proteins, where we have one protein labelled and the other
unlabelled. The kinetics are in the slow exchange regime on the chemical
shift timescale, but not in the slow exchange limit, so we see some
broadening of the signals during the titration. I was thinking of using
spectra from intermediate points in the titration (where there are both
bound and free signals) to estimate the association constant (by simply
integrating the signals to determine the concentrations of bound and free
labelled protein), but I suspect this might not be valid because of the
effects of exchange broadening on the resonances.
Could anyone tell me if my suspicions are valid, i.e. does the ratio of the
two peak volumes tell me the ratio of concentrations or not?
If not, I imagine i will need to fit the free and bound crosspeaks to a
model incorporating the exchange process (most free/bound pairs are
somewhat overlapped). It seems to me that this will be somewhat tricky,
given that the rate constant for complex formation is a second order one.
Does anyone know of software that is available to do this sort of analysis
of exchanging systems monitored by 2D NMR?
thanks in advance for any advice,
cheers,
Joel Mackay
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Dr Joel Mackay ph +61-2-9351-3906
ARC Research Fellow fax +61-2-9351-4726
Department of Biochemistry
University of Sydney
NSW 2006 Australia
http://www.biochem.usyd.edu.au/~joel/
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