Analytical Ultracentrifugation for Complex Systems

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APPLICANT'S DESCRIPTION: The characterization of the interactions between molecules is central to our understanding of both normal and pathological biochemistry. Consequently, the accurate description of molecular interactions plays a critical role in the development of safe and effective drugs. Improvements in the instruments and methods for characterizing molecular interactions are necessary for researchers to efficiently advance basic medical science. Analytical ultracentrifugation is one of the most rigorous, powerful and adaptable methods for determining the strength, stoichiometry and size of molecular complexes, but is currently limited to the analysis of relatively simple biochemical systems. With further development, analytical ultracentrifugation would be applicable to problems that are far more complex than can be investigated using current technology. The overall goal is to bring the power and rigor of analytical ultracentrifugation to bear on problems of interest in molecular and cellular biology. Collaborative efforts have been established with several NIH-funded researchers who use analytical ultracentrifugation in their work and who require the advances provided by the three specific aims of this proposal: 1) Development of rapid-scan absorbance optics. A photodiode-array-based detector capable of providing concentration profiles in < 1 s and at a 10 um radial resolution will be developed. Wavelength selection will require < 1 S with a reproducibility better than +/- 1 nm. These improvements will allow the analysis of rapidly-sedimenting boundaries at multiple wavelengths. 2) Development of radiolabel and Immuno-chemiluminescence detectors. Cell windows and centerpieces will be developed to permit the use of a luminescence detector for radiolabel and immuno-chemiluminescence detection. The specificity and sensitivity will allow the characterization of molecules under conditions approaching those found in vivo. The new centerpieces will decrease sample volume by up to 40-fold, while increasing the number of samples per experiment up to 4-fold, making it possible to rapidly and effectively conduct experimental protocols involving a large number of variables. 3) Development of an integrated ultracentrifuge operating system. A new instrument operating system will be developed to make the instrument easier to operate and improve the efficiency of data collection and analysis by logically organizing data acquisition procedures.