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Issue 8, 2016
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Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy

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Abstract

A large number of crystal structures of bacteriophage T4 lysozyme (T4-L) have shown that it contains two subdomains, which can arrange in a compact conformation (closed state) or, in mutants of T4-L, more extended structures (open state). In solution, wild-type T4-L displays only a single set of nuclear magnetic resonance (NMR) signals, masking any conformational heterogeneity. To probe the conformational space of T4-L, we generated a site-specific lanthanide binding site by attaching 4-mercaptomethyl dipicolinic acid via a disulfide bond to Cys44 in the triple-mutant C54T/C97A/S44C of T4-L and measured pseudocontact shifts (PCS) and magnetically induced residual dipolar couplings (RDC). The data indicate that, in solution and in the absence of substrate, the structure of T4-L is on average more open than suggested by the closed conformation of the crystal structure of wild-type T4-L. A slightly improved fit was obtained by assuming a population-weighted two-state model involving an even more open conformation and the closed state, but paramagnetic relaxation enhancements measured with Gd3+ argue against such a conformational equilibrium. The fit could not be improved by including a third conformation picked from the hundreds of crystal structures available for T4-L mutants.

Graphical abstract: Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy

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Supplementary files

Article information


Submitted
23 Nov 2015
Accepted
08 Dec 2015
First published
08 Dec 2015

Phys. Chem. Chem. Phys., 2016,18, 5850-5859
Article type
Paper
Author version available

Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy

J. Chen, Y. Yang, L. Zhang, H. Liang, T. Huber, X. Su and G. Otting, Phys. Chem. Chem. Phys., 2016, 18, 5850
DOI: 10.1039/C5CP07196H

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