Excitation energy transfer studies on the proximity between SH1 and the adenosinetriphosphatase site in myosin subfragment 1

Abstract

Excitation energy transfer studies were carried out to determine the distance between the ATPase site and a unique fast-reacting SH (referred to as SH1) in rabbit skeletal muscle myosin subfragment 1. The fluorescent moiety of the probe N-(iodoacetyl)-N''-(5-sulfo-1-naphthyl)ethylene-diamine was used as the donor attached at SH1. The chromophoric nucleotide analog 2''(3'')-O-(2,4,6-trinitrophenyl)ADP was used as the acceptor noncovalently bound at the ATPase site. The energy transfer efficiency was 56%, found by measuring the decrease in donor fluorescence lifetime. The critical transfer distance, R0(2/3), was 40.3 .ANG.. Since both donor and acceptor are likely to be rigidly attached, a statistical interpretation of the data was applied to determine distances. The method yielded the following conclusions: most probable distance = 38.7 .ANG.; maximum possible distance = 52 .ANG.; 10% probability for the distance to be < 20 .ANG.; 3% probability to be < 15 .ANG.. Despite the great influence that the 2 sites exert on each other, it is not likely that SH1 interacts directly with the ATPase site in myosin subfragment 1.