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Characterization of salt bridges to lysines in the protein G B1 domain

Tomlinson, R.A., Ullah, S., Hansen, P.E. and Williamson, M.P. (2009) Characterization of salt bridges to lysines in the protein G B1 domain. Journal of the American Chemical Society, 131 (13). pp. 4674-4684. ISSN 0002-7863

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Abstract

NMR investigations have been carried out on the B1 domain of protein G. This protein has six lysine residues, of which three are consistently found to form surface-exposed salt bridges in crystal structures, while the other three are not. The Nζ and Hζ chemical shifts of all six lysines are similar and are not affected significantly by pH titration of the carboxylate groups in the protein, except for a relatively small titration of K39 Nζ. Deuterium isotope effects on nitrogen and proton are of the size expected for a simple hydrated amine (a result supported by density functional theory calculations), and also do not titrate with the carboxylates. The line shapes of the J-coupled 15N signals suggest rapid internal reorientation of all NH3+ groups. pKa values have been measured for all charged side chains except Glu50 and do not show the perturbations expected for salt bridge formation, except that E35 has a Hill coefficient of 0.84. The main differential effect seen is that the lysines that are involved in salt bridges in the crystal display faster exchange of the amine protons with the solvent, an effect attributed to general base catalysis by the carboxylates. This explanation is supported by varying buffer composition, which demonstrates reduced electrostatic shielding at low concentration. In conclusion, the study demonstrates that the six surface-exposed lysines in protein G are not involved in significant salt bridge interactions, even though such interactions are found consistently in crystal structures. However, the intrahelical E35−K39 (i,i+4) interaction is partially present.

Item Type: Article
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > School of Biological Sciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield)
Depositing User: Miss Anthea Tucker
Date Deposited: 19 May 2009 11:12
Last Modified: 19 May 2009 11:12
Published Version: http://dx.doi.org/10.1021/ja808223p
Status: Published
Publisher: American Chemical Society
Refereed: Yes
Identification Number: 10.1021/ja808223p
URI: http://eprints.whiterose.ac.uk/id/eprint/8598

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