Fluorescence energy transfer in myosin subfragment-1

Abstract

Fluorescent probes were selectively introduced into skeletal muscle myosin subfragment-1 and the fluorescence emission characteristics of the labeled products studied. The fluorophores employed were the thiol-specific reagents N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, the spectral properties of which render them a particularly effective donor-acceptor pair in Forster energy-transfer studies. Alkali 1 L chain, labeled at a single cysteine with either of these probes, was incorporated into chymotryptic subfragment-1. The resultant, fluorescently labeled subfragment-1 was isolated by ion-exchange chromatography. Determination of the extent of incorporation by extinction and fluorescence indicated that greater than 80% of the subfragment-1 population possessed a fluorescently labeled alkali 1 L chain. The introduction of labeled alkali 1 did not perturb the K+-, Ca2+, or actin-activated ATPases of subfragment-1. The addition of ATP, liganded by various cations, to this singly labeled subfragment-1 induced at 6-10% decrease in the fluorescence intensity of the extrinsic chromophore. An intensity decrease of .apprx. 4% was obtained when the hydrolysis of ATP was complete and also upon direct addition of ADP. The ATP analogue adenylyl imidodiphosphate induced a decrease of .apprx. 7% in intensity. The addition of F-actin to the subfragment-1 in the presence of MgATP elicited no further fluorescence intensity change. A 2nd, appropriate fluorophore was introduced into the singly labeled subfragment-1 at the SH1 thiol on the H chain. Forster energy transfer was observed between this labeled site and the fluorophore previously introduced on the alkali 1 L chain. The measured efficiency of energy transfer indicated that the 2 fluorophores were .apprx. 40 .ANG. apart. The same value was obtained upon reversal of the donor and acceptor attachment sites, suggesting that the uncertainty in the calculated distance introduced by the choice of orientation factor is probably less than 20%. Steady-state observations did not reveal any obvious change in this distance upon the addition of MgATP and then F-actin to the doubly labeled subfragment-1.