Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded 15N/19F-labeled unnatural amino acid

SH3 is a ubiquitous domain mediating protein-protein interactions Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain Here, an orthogonal amber tRNA/tRNA synthetase pair for N-15/F-19-trifluoromethyl-phenylalanine (N-15/F-19-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites One-dimensional solution NMR spectra of backbone amide (N-15)H-1 and side-chain F-19 were obtained for SH3 with three different site-specific labels Site-specific backbone amide (N-15)H-1 and side-chain F-19 chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes (C) 2010 Elsevier Inc All rights reserved