How x-ray diffraction with synchrotron radiation got started

The need to record low angle scattering x-ray fibre diagrams from muscle with milli-second time resolution drove the use of synchrotron radiation as an x-ray light source. The first smudgy diffraction patterns were obtained from a slice of insect flight muscle. Out of this grew the EMBL Outstation at DESY.

Atomic structures give insight

The structure of the actin monomer and of the actin filament have been solved by protein crystallography (Kabsch et al ., 1990) and by x-ray fiber diffraction (Holmes et al ., 1990) . The crystal structure of the myosin subfragment 1 (Rayment et al ., 1993b) showed the myosin cross-bridge to have an extended C-terminal neck which looked like the anticipated lever arm and, moreover, a lever arm which was in the correct orientation and position to function as a lever arm (Rayment et al ., 1993a). In the last year a number of independent experiments provide results which are in excellent accord with the idea that the C-terminal tail functions as a lever arm and indeed provide evidence that it can move (see review (Holmes, 1997)). Furthermore, new crystal structures (Fisher et al ., 1995; Smith & Rayment, 1996a) with analogs of ATP bound appear to show an alternative orientation of the lever of the anticipated kind.

The crystallographic studies cited show two distinct structural states for the myosin cross-bridge: the "open" or "end" conformation which is characterized by the absence of nucleotide (rigor); and the "closed" or "beginning" state, which is favored by binding ATP or the products complex (ADP.Pi) (Fig 7). Myosin transports actin by switching between these two states. "Open" and "closed" refer to the status of the ATP binding site. This in turn is coupled to the rotation of the C-terminal lever arm. In the "closed" form the lever arm is at the beginning of the power stroke whereas in the "open" form it is at the end of the power stoke. The preference for "open" or "closed" is also controlled by binding to actin. It is likely that the closed state binds only weakly to actin. On this basis the structural states can be correlated with the Lymn-Taylor cycle.

Starting from an actin-myosin complex at the end of the power stroke, the binding of ATP brings about rapid closure of the ATP binding site and concomitant release from actin. The closed state hydrolyses ATP to ADP. Pi without attaching to actin. Thereafter, the rebinding of myosin in the closed or "beginning" conformation of the products complex to actin opens the site to facilitate release of the__-phosphate. Release of phosphate in turn induces an isomerisation to the open "end" conformation since it is the presence of the__-phosphate which stabilizes the closed form. The isomerisation results in large changes of angle of the "lever arm" (at the distal part of the myosin head). Since the S1 is strongly attached to actin at this stage this results in a 12nm transport of actin past myosin.

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