Our goal is to develop and apply an instrument for observing msec transients in phosphorescence anisotropy, induced by laser photolysis of caged ATP in ATP-dependent enzymes labeled with phosphorescent dyes. These experiments will provide direct information about nanosecond and microsecond molecular dynamics coupled with enzyme action. The first stage of this project is to develop an instrument that monitors polarization anisotropy of phosphorescence continuously with high time- resolution. This requires a dual-channel detection system with two polarizers and a real-time calculation of anisotropy. In addition, in order to detect selectively the (long-lived) phosphorescence, rejecting the (short-lived) fluorescence, we will use a high-frequency pulsed laser light source (a cavity-dumped argon ion laser) and gated detection. This selectivity for phosphorescence will be augmented by using wavelength-selective interference filters. In order to obtain the high signal-to-noise necessary for msec time-resolution, we will use """"""""structured light"""""""" techniques to focus the polarized laser beam precisely on the sample. To produce a large transient increase in the ATP concentration, the sample will be bathed in caged ATP, a photolabile ATP precursor, and the sample will be illuminated by a giant UV pulse from a XeCl excimer laser. The samples used to test and apply this new technology will be either muscle fibers, labeled with eosin-maleimide on the myosin cross-bridge, or muscle membranes, labeled with erythrosin iodoacetamide attached to the Ca-pumping ATPase. Protein rotational motions have been proposed to play important roles in these and other energy-transducing enzymes, and this technology will make possible a whole new class of biophysics experiments, in which nanosecond and microsecond molecular dynamics can be monitored directly with high sensitivity and time-resolution during the transient phase of enzymatic activity, thus helping to elucidate the molecular mechanisms of fundamental physiological processes.
| Tanner, J W; Thomas, D D; Goldman, Y E (1992) Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres. J Mol Biol 223:185-203 |
| Fajer, P G; Fajer, E A; Schoenberg, M et al. (1991) Orientational disorder and motion of weakly attached cross-bridges. Biophys J 60:642-9 |
