This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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, troponin C, and human centrin 2).
Showing the most recent 10 out of 696 publications
