A Glimpse at Loop 1 Movement in Smooth Muscle Using Intrinsic Tryptophan Fluorescence

Abstract

Smooth muscle myosin has two N-terminal isoforms that result from alternative splicing of loop 1. Loop 1 contains a seven amino acid insert (QGPSFSY) in one isoform (SM-B) that is absent in the other (SM-A). It has been shown that the presence of the insert causes a two-fold increase in the rate of in-vitro actin sliding velocity and actin-activated ATPase (Rovner et al., J. Muscle Res. Cell Motil. 18:103, 1998). Based on these results and its proximity to the active site it was hypothesized that loop 1 plays a role in modulating the release of ADP (Spudich, Science 372:515, 1994). However, little is known about the conformation of loop 1 in different nucleotide states, as it is absent in crystal structures. To examine the position of loop 1 and its potential role in ADP release we have engineered a single tryptophan residue into loop 1 at position 215. Using the intrinsic tryptophan fluorescence from W215 and fluorescent analogs of ADP and ATP we have looked at the position of loop 1 as a function of temperature. The results suggest two conformations of loop 1 in the ADP state, both an open and closed form. The distance between loop 1 and the active site decreases for both nucleotides from 25-15°C. At 10 °C loop 1 moves away from the active site in the ADP state, while there is no additional movement in the presence of ATP. This is the first data to demonstrate movements of loop 1 associated with different nucleotide states of the myosin active site, giving insight into how it may contribute to nucleotide release.