"Proteomics, Chapter IV: Mass Spectrometry Gives
Affinities and Conformational Changes in Protein-Ligand Interactions"
Michael L. Gross
Departments of Chemistry, Medicine, and Immunoloy, Washington University
in St. Louis, St. Louis, Mo 63130 USA
Protein-ligand binding and the concomitant conformational change in
the protein are of crucial importance in biophysics and drug design.
We recently described in a 2003 communication to JACS a novel method
to quantify Protein-Ligand Interactions in solution by Mass Spectrometry,
Titration and H/D Exchange (PLIMSTEX). The approach can determine the
conformational change, binding stoichiometry and affinity in protein-ligand
interactions including those that involve small molecules, metal ions,
and peptides. Binding constants obtained by PLIMSTEX for four model
protein-ligand systems agree with K values measured by conventional
methods. At higher protein concentration, the method can be used to
determine quickly the binding stoichiometry and possibly the purity
of proteins. Taking advantage of concentrating the protein on-column
and desalting, we are able to use different concentrations of proteins,
buffer systems, salts, and pH in the exchange protocol. High picomole
quantities of proteins are sufficient, offering significantly better
sensitivity than that of NMR and X-ray crystallography. Automation could
make PLIMSTEX a high throughput method for library screening, drug discovery,
and proteomics. For example, we expect that PLIMSTEX will be useful
for evaluation of the binding of small-molecule drug candidates to target
proteins. The approach is being expanded to peptide/peptide and protein/protein
interactions by using gramicidin and insulin as examples. The lecture
will cover the basic principles of PLIMSTEX and associated kinetic measurements
and its application to ligand binding to calmodulin, fatty-acid binding
protein, and self association of gramicidin and insulin as model systems.
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