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 (15)N/(19)F-trifluoromethyl-phenylalanine ((15)N/(19)F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide ((15)N)(1)H and side-chain (19)F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide ((15)N)(1)H and side-chain (19)F 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.
Shi
Pan
P
National Laboratory for Physical Science at Microscale, University of Science and Technology of China, Hefei, Anhui 230026, PR China.
Xi
Zhaoyong
Z
Wang
Hu
H
Shi
Chaowei
C
Xiong
Ying
Y
Tian
Changlin
C
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D005461:Fluorine / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling; D009124:Muscle Proteins; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D000596:Amino Acids; D009124:Muscle Proteins; D009587:Nitrogen Isotopes; C486523:SORBS3 protein, human; D005461:Fluorine; D010649:Phenylalanine