The study of the molecular mechanisms used by cells for the assembly of membranes is the principal focus of the instrumentation being developed in this project. The lipid bilayer of membranes has been shown to self-assemble at a critical point, T*, which is identical to the physiological temperature, previously measured by equilibrium thermodynamic methods. Evaluation of T* under equilibrium conditions has been limited by the intrinsic slowness of the bilayer assembly processes at the critical point. Thus a measurement of T* is limited by the speed at which equilibrium is reached, and may take several weeks to complete. We have developed a scanning method for measuring T* that obviates the need to wait for equilibrium and allows a complete T* measurement in several hours. A commercial spectrofluorometer is being modified to control the temperature in the sample solution to +/- 0.01C. The system incorporates precision miniature thermistors, peltier devices, and feedback control systems to individually control the temperature in four cuvettes. In addition, each cuvette is fitted with a speed controlled stirring motor to maintain the lipid solution in suspension. The entire system, control and data acquisition, is managed by a personal computer via interface cards and custom software written for this application. This method is now being tested on lipids with known critical temperatures, and the specimen preparation techniques are being refined to maximize the sample reproducibility. This is a continuation of Intramural Research Project Z01-RR-10467-03 BEI.
