[RASMB] Weak Ligand Binding by DSC

[John Sumida]

Dear RASMB,

 

We are working with a controlled substance binding to protein target - I
apologize for being vague but disclosure rules limit me from being more
specific. The affinity estimate by yeast expression is very approximately
100uM Kd and the real affinity is quite possibly weaker.   

 

The nature of the ligand and its toxicity preclude its use in the AUC due to
the potential to aerosolize the material

 

Additionally, the controlled drug material interacts with a Series S CM5
chip likewise make quantitation by SPR difficult.

 

For ITC, the given interaction would require 1mM protein in the calorimetric
cell, which is not feasible for us.  One could design a displacement ITC
experiment with a suitable strong endothermic binder but that would also not
be practical - at least not yet. 

 

I have been considering using DSC to monitor the shift in Tm (Brandts et.
al. Biochem. Vol.24, 1990) and extract the Kd at the Tm; we would have to
correct the Kd to room temperature using the enthalpy of binding determined
by a reverse titration using the ITC instrument.  I know that this approach
has been useful in the measurement of ultra-tight binding, (Gomez et al. JMB
vol. 252, 1995), and if my reading of some of reviews in the literature is
accurate it should also be useful for the quantitation of weak affinity
interactions.  However I have not found too many examples of using this
approach to quantitate weak affinity interactions.  The advantage of this
approach seems to me is that it would only require 2uM protein and that the
interaction is saturated.

 

I wonder if anyone knows if ligand binding by DSC has been used to
quantitate weak interactions and if so if someone might be kind enough to
point me at a reference.

 

I very much appreciate any comments and advice that might be provided, and
thank you all in advance for your time.

 

Best regards,

John Sumida,

 <http://depts.washington.edu/cidb4bio/index.shtml> Analytical Biopharmacy
Core Facility

University of Washington

 

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