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The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X‐ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH‐values. The observed light‐induced intensity changes of in‐plane X‐ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X‐ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3–3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at ‐180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.

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Lanyi (1990Lanyi ( , 1991a postulated an irreversible transition the FTIR spectra occur between M 2 and M G . This between M 1 and M 2 . This could not, however, hitherto suggests, that the tertiary structural changes between be unambiguously proven experimentally, although the M 1 and M 2 are responsible for the switch opening the presence of two M intermediates (M 1 and M 2 ) had been cytoplasmic half-channel of BR for reprotonation to deduced much earlier from spectroscopic measurements complete the catalytic cycle. These tertiary structural (Korenstein et al., 1978). changes seem to be triggered by a charge redistribution UV-VIS (Varo and Lanyi, 1991b;Varo et al., 1992; which might be a common feature of retinal proteins Zimanyi et al., 1992) and Fourier transform infrared also in signal transduction.(FTIR) spectroscopy (Ormos, 1991;Perkins et al., 1992) Keywords: conformational changes/hydration/ revealed a transition from M 1 to M 2 by a slight shift in M intermediates/photocycle/proton pumping the absorption maximum of the M intermediate and changes in the amide-I region (1650-1670 cm -1 ), respectively. Later, the FTIR results were, however, re-interpreted as giving no evidence for a M 1 to M 2 transition, due to

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Low-angle x-ray diffraction diagrams have been recorded from frog sartorius muscles by using synchrotron radiation as a high-intensity x-ray source. This has enabled changes in some of the principal reflections of interest to be followed with a time resolution of 1ims, during small but very rapid length changes imposed on a contracting muscle. The 143-A meridional reflection, which is believed to arise from a repeating pattern ofmyosin crossbridges along the length of the muscle, shows large changes in intensity in these circumstances. During both rapid releases and rapid stretches, by amounts that produce a translation ofactin and myosin filaments past each other by about 100 A and that are completed in about a millisecond (i.e., before significant cross-bridge detachment would be expected), an almost synchronous decrease in 143-A intensity occurs, by 50% or more. This is followed, in the case ofquick releases, by a rapid partial recovery of intensity lasting 5-6 ms (which may represent cross-bridge release and reattachment) and then by a more gradual return to the normal isometric value. Quick stretches show only the slower return of intensity. Immediately after the length change, the initial drop in 143-A intensity can be reversed if the release-(or stretch) is reversed. These changes provide evidence ofa more direct kind than has hitherto been available that the active sliding ofactin filaments past myosin filaments during contraction is produced by longitudinal movement of attached cross-bridges.The outstanding problem in understanding the mechanism of muscular contraction is to discover the nature of the process by which the relative sliding force between actin and myosin filaments is developed. It is generally supposed that the crossbridges, the enzymatically active heads of the myosin molecules, function in a cyclical manner to produce this force. It is thought that they attach to actin in one part of their cycle, then undergo some structural change that enables them to exert tension and, during shortening, to pull the actin along a short distance-probably 100 A or so-towards the center ofthe A-band. They then detach from actin and are recharged by ATP before beginning a new cycle of attachment further out along the actin filament (1, 2). Whilst this general mechanism has been able to give a good account of many phenomena in muscle, it has proved remarkably difficult to produce direct and decisive evidence that the cross-bridges really do behave in this fashion, because of. the inherent difficulty of obtaining dynamic structural information on the size and time scales involved. Moreover, because the processes take place asynchronously at all the cross-bridges, probes of their configuration will usually give only an averaged value of changes in them during the cycle.Surviving muscles give low-angle x-ray diffraction diagrams that contain a considerable amount of information about.-the configuration ofthe cross-bridges and that alter incharacteristic ways during contraction (3-7). The changes in t...

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