Guignard, Laurent; Ozawa, Kiyoshi; Pursglove, Sharon; Otting, Gottfried; Dixon, Nicholas
A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl-prolyl cis-trans isomerase PpiB was expressed in this system with dual amino acid-selective isotope labeling to identify the NMR signal from the active site-residue Arg87. Addition of the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide selectively shifted its15N-HSQC cross peak, confirming binding to the active site. As...[Show more]
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