A novel restricted photoaffinity spin-labeled non-nucleoside ATP analogue as a covalently attached reporter group of the active site of Myosin subfragment 1

The photoaffinity spin-labeled non-nucleoside ATP analogue, 2-(4-azido-2-nitrophenyl)amino-2,2-(1-oxyl-2,2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene)ethyl triphosphate (SSL-NANTP), has been shown to be a substrate for skeletal mysoin subfragment 1 (S1) that can be photoincorporated at the active site of S1 Chen, X., et al. (2000) Bioconjugate Chem. 11, 725-733]. Electron paramagnetic resonance spectroscopy shows that the probe undergoes restricted motion with respect to the protein. The parent compound, NANTP (2-(4-azido-2-nitrophenyl)amino]ethyl triphosphate), is specifically photoincorporated at Trp-130 on the amino-terminal 23 kDa tryptic fragment in rabbit skeletal myosin. Surprisingly, amino acid sequence analysis shows that SSL-NANTP is photoincorporated on the carboxy-terminal 20 kDa tryptic fragment at Lys-681 on the side opposite Trp-130 in the nucleotide pocket. This is the first direct evidence showing that this residue in the 20 kDa tryptic fragment is close enough to the active site to be photolabeled by trapped ATP analogues. After actin treatment in the presence of MgATP, SSL-NANDP-labeled myosin S1 had normal ATPase activity, indicating that photolabeling did not significantly alter the enzymatic properties of S1. Photoincorporated SSL-NANDP was bound inside the nucleotide site of S1, with an effective concentration of 20 mM as judged by the concentration of MgADP needed to displace it. Molecular dynamics simulations suggest that the ability of NANTP and SSL-NANTP to photolabel different sites results from different orientations of the phenyl ring in the active site. For SSL-NANTP, the p-azido group on the phenyl ring points toward Lys-681. For NANTP, it points in the opposite direction toward Trp-130.