Synthesis and characterization of novel spin-labeled photoaffinity nonnucleoside analogues of ATP as structural and EPR probes for myosin

Two new spin-labeled photoreactive nonnucleoside ATP analogues, 1-(4-azido- 2-nitrophenyl)amino-3-( 1-oxyl-2,2,5,5-tetramethylpyrrolidinyl-3-carbamido) -2-propyl triphosphate (SL-NANTP) and 2-(4-azido-2-nitrophenyl)amino-2,2-(1 -oxyl-2 2,6,6-tetramethyl-4-piperidylidene)di(oxymethylene) ethyl triphosph ate (SSL-NANTP), were synthesized and characterized. This study aims to dev elop a second generation of NANTP-based analogues containing immobile spin labels that can be used to monitor conformational changes in myosin during the contractile cycle of muscle. Previous studies have shown that both a ph otoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] et hyl triphosphate (NANTP) [Nakamaye et al. (1985) Biochemistry 24, 5226-5235 ], and a photoaffinity ATP analogue, 3'(2')-O-4-[4-oxo-(4-amino-2,2,6,6-tet ramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5'-triphosphate (S L-Bz(2)ATP) [Wang et al. (1999) J. Muscle Res. Cell MotiL. 20, 749-753], be have like ATP in their interactions with myosin. Remarkably, photolabeled m yosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-198 7]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminome thyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. T he two new probes interact with myosin subfragment-1 (S1) in a manner analo gous to ATP, and after photoincorporation, labeled S1 recovers full activit y after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very h igh degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S 1, constrained to move within a cone of angle 52 degrees (full-width, half- max). Unlike the parent, NANTP, which photolabels on the 23 kDa tryptic fra gment of S1, SSL-NANTP photolabels on the 20 kDa fragment. Its highly immob ile nature means that it is potentially a useful reporter group to monitor cross-bridge motion in muscle fibers.