Prodan Fluorescence Reflects Differences in Nucleotide-Induced Conformational States in the Myosin Head and Allows Continuous Visualization of the ATPase Reactions

Abstract

The noncovalent fluorescent probe 6-propionyl-2-(dimethylamino)naphthalene (prodan) binds stoichiometrically to myosin subfragment-1 (S-1) without affecting the ATPase and actin-binding properties of S-1. Neither ATP nor actin interferes with the prodan binding. Free prodan exhibits a green emission peak at 520 nm. However, the prodan bound to S-1 and the S-1.ADP complex shows blue emission peaks at 460 and 450 nm, respectively, which allow easy separation of the fluorescence contributions from the free and bound probes. In the S-1.ADP.Pi state, the blue emission peak is further shifted to 445 nm with a large (4.5-fold) fluorescence enhancement. Thus, prodan in the presence of S-1 exhibits predominantly blue fluorescence only during ATP hydrolysis, and so visualizes the ATPase reaction continuously. The initial velocities of the steady state of the Mg2+-, Ca2+-, and actin-activated ATPases can be conveniently calculated from the blue fluorescence changes. The ability of different nucleoside triphosphates (NTP) to enhance the blue fluorescence of prodan follows the order ATP > CTP > UTP > ITP > GTP. This order agrees with those of the extent of hydrophobicity near the ribose of the corresponding nucleoside diphosphates (NDP) trapped to S-1 with orthovanadate (Vi) [Hiratsuka, T. (1984) J. Biochem. (Tokyo) 96, 155-162] and the ability of different NTPs to support force production in muscle fibers [Regnier, M., et al. (1993) Biophys. J. 64, A250]. The rate of formation of the corresponding S-1.NDP.Vi complex also follows this order, whereas the NTPase rate follows the reverse order. These results indicate that nucleotide-induced changes in prodan fluorescence correspond to the nucleotide-induced conformational states of S-1. Thus, the use of prodan in studies of the myosin ATPase offers a new and promising approach not only to monitoring the ATPase reaction but also to investigating the structural changes during ATP hydrolysis.

ATP-induced Opposite Changes in the Local Environments around Cys697 (SH2) and Cys707 (SH1) of the Myosin Motor Domain Revealed by the Prodan Fluorescence

“…On the other hand, the structure of the fluorophore moiety of BD, the 6-acyl-2-dimethylaminonaphthalene group, is the same as those of another thiol reagent AD and the noncovalent probe prodan (21,22). Thus BD can be expected to show fluorescence that is extremely sensitive to solvent polarity as well as prodan and AD (21)(22)(23)(24).…”

“…AD proved to be a very useful labeling reagent for SH2 because of its selectivity to SH2 and moderate reactivity. Furthermore, it has the same fluorophore as that of prodan, which has frequently been used as an extremely sensitive environmental probe for proteins (21)(22)(23)(24). These properties allow ready use of AD not only as a specific fluorescent label for SH2 but as a sensitive probe to precisely describe the conformational changes around SH2.…”

“…Unlike the vast majority of the more commonly used fluorophores, the noncovalent fluorescent probe prodan, the parent fluorophore of AD and BD (Scheme 1), should be particularly well suited to probe the surface of proteins by virtue of the exquisite sensitivity of the fluorophore to minor changes in the environment (21)(22)(23)(24). It gives a 130-nm shift in the emission maximum from 401 nm in cyclohexane to 531 nm in water (21).…”

Fluorescence

“…Its fluorescence is strongly quenched in aqueous solution and usually increases markedly and shifts from green to blue when the dye binds to a protein [102,103]. Similar shifts of emission with changes in the local environment occur with Prodan (6-propionyl-2-(dimethylamino)naphthalene) and related dyes [8,104,105]. Di-4-ANEPPS is one of a group of "voltage-sensitive" dyes that can be used to sense changes in membrane potentials in neurons and other electrically active cells.…”

Enantioselective synthesis and application of the highly fluorescent and environment-sensitive amino acid 6-(2-dimethylaminonaphthoyl) alanine (DANA)Electronic supplementary information (ESI) available: experimental details. See http://www.rsc.org/suppdata/cc/b2/b205224e/

“…3 PRODAN has been used as a probe in many biological systems, perhaps most notably in the study of lipid bilayers 4 and in proteins which have a fortuitous binding site for the fluorophore. 5 Derivatives of PRODAN have also been attached covalently to proteins via reaction with amino acid side chain thiols with 6-acryloyl-2-(dimethylamino)naphthalene 6,7 or 6-bromoacetyl-2-dimethylaminonaphthalene (BADAN). 8 These methods provided useful information about the macromolecule of interest but are limited to systems with uniquely reacting amino acid side chains.…”