ac051294f_si_001.xls (58.5 kB)
Probing Protein Ligand Interactions by Automated Hydrogen/Deuterium Exchange Mass Spectrometry
dataset
posted on 2006-02-15, 00:00 authored by Michael J. Chalmers, Scott A. Busby, Bruce D. Pascal, Yuanjun He, Christopher L. Hendrickson, Alan G. Marshall, Patrick R. GriffinAmide hydrogen/deuterium exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low
throughput of the technology has limited its impact on
drug screening and lead optimization. Automation increases the throughput of H/D exchange to make it
compatible with drug discovery efforts. Here we describe
the first fully automated H/D exchange system that
provides highly reproducible H/D exchange kinetics from
130 ms to 24 h. Throughput is maximized by parallel
sample processing, and the system can run H/D exchange
assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural
perturbations in the ligand-binding domain (LBD) of the
nuclear receptor PPARγ induced upon binding a full
agonist and a partial agonist. PPARγ is the target of
glitazones, drugs used for treatment of insulin resistance
associated with type II diabetes. Recently it has been
shown that partial agonists of PPARγ have insulin sensitization properties while lacking several adverse effects
associated with full agonist drugs. To further examine the
mechanism of partial agonist activation of PPARγ, we
extended our studies to the analysis of ligand interactions
with the heterodimeric complex of PPARγ/RXRα LBDs.
To facilitate analysis of H/D exchange of large protein
complexes, we performed the experiment with a 14.5-T
Fourier transform ion cyclotron resonance mass spectrometer capable of measuring mass with accuracy in the
ppb range.