This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We developed 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 consts. obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concn., 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 concns. 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 crystallog. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics. We are continuing the development of a protocol for H/D exchange of proteins. The protocol is directed at an aspect of protein folding that is not being pursued by others;namely the interaction of proteins with metal ions and with other substances such as proteins, peptides, drugs. An area of particular interest is the conformational changes that occur to calcium-binding proteins (e.g., calmodulin, calbinden, and human centrin 2). Another protein system of interest is fatty acid binding protein, an area in which scores of mutants are available from Dr. A. Kleinfeld at Torrey Pines Research Institute. A third system is DNA-binding proteins, and in this area we are working on the application of PLIMSTEX to hTRF2, a small protein for which binding off rates are competitive with rates of H/D exchange. This provides a challenge in modeling PLIMSTEX and kinetic data, and the modeling is underway.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-BPC-H (40))
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Washington University
Internal Medicine/Medicine
Schools of Medicine
Saint Louis
United States
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