Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: Evidence that the start of the crossbridge power stroke in muscle has variable geometry

The mechanism of binding of myosin subfragment-1 (S1) to actin in the absen ce of nucleotides was studied by a combination of stopped-flow fluorescence and ms time resolution electron microscopy, The fluorescence data were obt ained by using pyrene-labeled actin and exhibit a lag phase. This demonstra tes the presence of a transient intermediate after the collision complex an d before the formation of the stable "rigor" complex, The transient interme diate predominates 2-15 ms after mixing, whereas the rigor complex predomin ates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixi ng revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed th e "arrow-head" appearance characteristic of rigor. The most likely explanat ion of the disorder of the transient intermediate is that the binding is th rough one or more flexible loops on the surfaces of the proteins. The trans ition from disordered to ordered binding is likely to be part of the force- generating step in muscle.