On how a myosin tryptophan may be perturbed.

Abstract

A weil-known indication that a nucleotide has bound to myosin Is the enhancement of the fluorescence of a speciflc tryptophan in the "subfragment 1" segment of the protein. Empirically the effect has been enormously useful in myosin enzymology. But beyond an early suggestion that it aries from a purine-tryptophan charge-transfer complex, the mechanism of the effect has not been considered. Here we consider the alternative that it arises from an Ionizable group (either another residue or the phosphate of the nucleotide) whose proimit to the tryptophan is altered by substrate bnding. We study this possibility by studying the interaction of an ionizable residue and tryptophan when both are incorporated in a diketopiperazine structure. The geometry of the ituation is inferred from molecular mehaics simulations. Unexpectedly, the best explanation seems to be that the field of the imposed charge, acting across space, affects events in the excited state of the indole.The perturbation of the absorbance (1) and emission (2) (8), but alternative explanations have not been considered. We undertook the present study, hoping to get clues about the aforementioned effect in myosin and, more generally, information about environmental effects on tryptophan. Our specific goal was to study the effect of a single charge positioned in a known relation to the indole ring, using a setup in which the sign and quantity of the charge (and to a limited extent the geometry of the arrangement) could be varied. We sought to accomplish this goal by using diketopiperazines constructed from. tryptophan and the L or D forms of amino acids such as histidine or aspartic acid, using appropriate molecular mechanical analysis to characterize the charge-indole relation. MATERIALS AND METHODSPreparation of the Diketopiperazines. The strategy followed was to prepare t-butoxycarbonyl (Boc)-protected amino acids, to couple these into linear dipeptide esters, and then to cyclize them and remove the protective groups.Boc-Trp and Boc-Asp(13-OBz) (where Bz = benzoyl) were prepared according to Morodes et al. (9). Glu(-OBz) OBz-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6791TosOH and His-OMe*2HCl were prepared according to Zervas et al. (10) and Humphlett and Wilson (11), respectively. Linear protected dipeptide esters were prepared by the N,N'-dicyclohexylcarbodiimide/N-hydroxysuccinimide method (12). Dimethylformamide was the solvent for coupling. The products were purified from ethyl acetate petroleum ether, except Boc-L-Trp-L-His-OMe, which was purified from warm ethanol. In TLC, each of the products gave a single spot (ninhydrin negative) using the KI/toluidine reaction for detection (13). To prepare the diketopiperazines, a Boc group was removed by dissolving 10 mmol of Bocprotected dipeptide ester in 120 ml (240 ml for the histidinecontaining dipeptide) of 0.1 M HCl/96% formic acid...

X-ray Structure of the Magnesium(II)·ADP·Vanadate Complex of the Dictyostelium discoideum Myosin Motor Domain to 1.9 Å Resolution,

“…The exact reason for the fluorescence enhancement cannot be explained by the present structures since it is likely that the exact nature of the conformational change that occurs around Trp 501 is influenced by the essential light chain which is missing from the structures of S1Dc. It has been suggested that the fluorescence enhancement might be due to an interaction between an ionizable side chain and the indole ring (Bivin et al, 1993). The use of the ADP,Vi complex to account for the fluorescence enhancement must also be qualified by the observation that in rabbit skeletal muscle myosin the ADP,Vi complex shows a lower level of 8 were prepared with the programs FRODO and MOLVIEW (Jones, 1985;Smith, 1993).…”

Isolating and Localizing ATP-Sensitive Tryptophan Emission in Skeletal Myosin Subfragment 1

“…Isolation of ASTs Other than Trp510. Trp510 is a known AST (9)(10)(11)(12)29). The possibility that the ASTs include residues other than Trp510 was considered using 5′IAF to specifically modify SH1 in S1.…”

Mechanism for Coupling Free Energy in ATPase to the Myosin Active Site

“…Tryptophan emission intensity from S1 is altered by ATP binding to its active site (Werber et al, 1972). Trp510 is one of the ATP-sensitive tryptophans (Johnson et al, 1991;Hiratsuka, 1992;Bivin et al, 1993;Papp & Highsmith, 1993;Park et al, 1996b,c). The nucleotide-sensitive tryptophan emission showed that the stable complexes of ADP with vanadate, beryllofluoride, or aluminofluoride bound to S1 mimic ATPase intermediates (Werber et al, 1992).…”