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NMR methods for the study of protein electrostatics

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Title NMR methods for the study of protein electrostatics
Period 05 / 2007 - unknown
Status Current
Research number OND1329068
Data Supplier Website RUG

Abstract

In collaboration with the group of Prof. Linse (Lund University, Sweden) new NMR pulse schemes have been devised to determine the protonation constants of individual amino acid side chains in small proteins with sequence specificity. Novel indirect 13C and 15N NMR techniques were developed that are suited for the pKa determination of lysine and arginine side-chains. Such methods supersede commonly employed 1H-based NMR techniques in terms of accuracy, sensitivity and selectivity. Because of the increased reliability these methods are expected to be of widespread use for the study of electrostatics in proteins, and may serve as benchmarks for computational methods. Individual aspartate and glutamate side chain carboxylate pKas were determined from 13C' chemical shift data for a variant of the B1 domain of protein G. Chemical shift titration curves appear skewed from "ideal" behavior and we have developed a framework for the analysis of these skewed curves, based on local charge-charge interactions and electrostatic coupling. It is established that differences in self-energy, and not direct charge-charge interactions, are responsible for shifted pKa values.

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Project leader Dr. F.A.A. Mulder

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