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 triphosphate (SSL-NANTP), were synthesized and characterized. This study aims to develop 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 photoaffinity nonnucleoside ATP analogue, 2-[(4-azido-2-nitrophenyl)amino] ethyl 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-tetramethyl-piperidino-1-oxyl)-4-benzoyl] benzoyl adenosine 5'-triphosphate (SL-Bz(2)ATP) [Wang et al. (1999) J. Muscle Res. Cell Motil. 20, 743-753], behave like ATP in their interactions with myosin. Remarkably, photolabeled myosin recovers all of its normal enzymatic properties after treatment with actin in the presence of MgATP [Luo et al. (1995) Biochemistry 34, 1978-1987]. For SL-NANTP, the spin label moiety is attached to NANTP via an aminomethyl side chain. In SSL-NANTP, attachment is via a restricted spiro ring. The two new probes interact with myosin subfragment-1 (S1) in a manner analogous to ATP, and after photoincorporation, labeled S1 recovers full activity after treatment with actin and MgATP. The electron paramagnetic resonance (EPR) spectrum resulting from S1 photolabeled with SL-NANTP shows a very high degree of probe mobility. However, the EPR spectrum of S1 photolabeled with SSL-NANTP shows that the probe is highly immobilized with respect to S1, 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 fragment of S1, SSL-NANTP photolabels on the 20 kDa fragment. Its highly immobile nature means that it is potentially a useful reporter group to monitor cross-bridge motion in muscle fibers.
Chen
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School of Molecular Biosciences, Department of Pure and Applied Mathematics, Department of Chemistry,Washington State University, Pullman, Washington 99164, USA.
Grammer
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Cooke
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Pate
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Yount
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D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry*; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D015879:Myosin Subfragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels; D014638:Vanadates
D000199:Actins; D000345:Affinity Labels; D001386:Azides; D007202:Indicators and Reagents; D015879:Myosin Subfragments; D013113:Spin Labels; D014638:Vanadates; D000255:Adenosine Triphosphate; C047568:2-((4-azido-2-nitrophenyl)amino)ethyl triphosphate; D009218:Myosins