title: Interaction of myosin subfragment 1 with fluorescent ribose-modified nucleotides. A comparison of vanadate trapping and SH1-SH2 cross-linking.
abstract: The environment near the ribose binding site of skeletal myosin subfragment 1 (S1) was investigated by use of two adenosine 5'-diphosphate analogues with fluorescent groups attached at the 2'- and 3'-hydroxyls of the ribose ring. We have compared steady-state and time-resolved fluorescent properties of the reversibly bound S1-nucleotide complexes and the complexes generated by N,N'-p-phenylenedimaleimide (pPDM) thiol cross-linking or vanadate (Vi) trapping. A new fluorescent probe, 2'(3')-O-[N-[2-[[[5-(dimethylamino)naphthyl]sulfonyl] amino]ethyl]carbamoyl]adenosine 5'-diphosphate (DEDA-ADP), which contains a base-stable carbamoyl linkage between the ribose ring and the fluorescent dansyl group, was synthesized and characterized. For comparison, we performed parallel experiments with 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (MANT-ADP) [Hiratsuka, T. (1983) Biochim. Biophys. Acta 742, 496-508]. Solute quenching studies indicated that both analogues bound reversibly to a single cleft or pocket near the ribose binding site. However, steady-state polarization measurements indicated that the probes were not rigidly bound to the protein. The quantum yields of both fluorophores were higher for the complexes formed after trapping with pPDM or Vi than for the reversibly bound complexes. Both DEDA-ADP and MANT-ADP, respectively, had nearly homogeneous lifetimes free in solution (3.65 and 4.65 ns), reversibly bound to S1 (12.8 and 8.6 ns), and trapped on S1 by pPDM (12.7 and 8.7 ns) or Vi (12.8 and 8.6 ns). In contrast to the quantum yields, the lifetimes were not increased upon trapping, compared to those of the reversibly bound states. These results suggested that static quenching in the reversibly bound complex was relieved upon trapping. Taken together, the results suggest that there was a conformational change near the ribose binding site upon trapping by either pPDM or Vi. On the basis of the quantum yield, lifetime, polarization, and solute accessibility studies, we could not detect differences between the S1-pPDM-nucleotide analog complex and the S1-Vi-nucleotide analogue complex for either analogue. Thus, previously observed differences with the adenine modified nucleotide analogue 1,N6-ethenoadenosine diphosphate (epsilon ADP) could not be detected with these ribose-modified probes, indicating that structural differences may be localized to the adenine binding site and not transmitted to the region near the ribose ring.
authors.affiliation: Department of Chemistry, Colorado College, Colorado Springs 80903.
authors.forename: C R
authors.initials: CR
authors.lastname: Cremo
authors.forename: J M
authors.initials: JM
authors.lastname: Neuron
authors.forename: R G
authors.initials: RG
authors.lastname: Yount
mesh_terms: D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D009682:Magnetic Resonance Spectroscopy; D015879:Myosin Subfragments; D011817:Rabbits; D012266:Ribose; D013438:Sulfhydryl Compounds; D014638:Vanadates
chemical_list: D003432:Cross-Linking Reagents; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D013438:Sulfhydryl Compounds; D014638:Vanadates; D012266:Ribose