TIME RESOLVED XRAY DIFFRACT: ULTRATHIN LANGMUIR BLODGETT & SARCOPLASMIC RETICUL

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The Ca2+-ATPase of the sarcoplasmic reticulum (SR) in skeletal and cardiac muscle is a prototypical energy-dependent ion transport ATPase. We are seeking to elucidate the details of the molecular mechanism responsible for the energy-dependent transport of calcium ions using time resolved resonance and non-resonance diffraction studies of the profile structure of the Ca2+-ATPase within hydrated oriented multilayers of isolated sarcoplasmic reticulum and vectorially-oriented single monolayers of the detergent-solubilized Ca2+-ATPase. Early experiments using bending magnet sources at the NSLS were limited to time scales of longer than 400 milliseconds. Initial time-resolved studies at the BioCAT doubly-focused undulator-magnet beamline at the APS/ANL focused on determining the minimum time-frame that could be recorded while still provide data of high statistical accuracy. The minimum duration of the in-line shutter was 5 milliseconds. The full available flux saturated a FUJI Image Plate in a 5 millisecond exposure. X-ray diffraction patterns of good statistical quality were recorded from ultra-thin Langmuir-Blodgett (LB) multilayers films by attenuating the beam intensity by 100-fold. These tests indicated that there was sufficient flux to record 50 microsecond exposures using the full available flux. Subsequent studies used the 16-channel prototype of the Multi-Element Detector (MED) which is capable of recording contiguous time frames of 16 microseconds duration with 150 nanosecond blind time between frames. The detectors capabilities were demonstrated by recording 1024 contiguous time-frames of 15 millisecond duration form a 2-bilayer Cd-Arachidate LB-flim. Time-resolved x-ray diffraction data were recorded from Ca2+-ATPase multilayers in contiguous time-frames of 150 millisecond duration. Flash photolysis of caged-ATP was used to synchronize the ensemble.