The intent of this project is to develop new and improve existing instrumentation and to develop new experimental and data analysis methods for the characterization of biological macromolecules and the study of their interactions. Several new methods have been developed for the analysis of data from analytical ultracentrifugation. The first development is an extension of last year's major breakthrough in the use of light intensity data rather than light absorbency data from the analytical ultracentrifuge. The intensity data is advantageous because of its superior statistical properties and its potential to provide better parameter estimates in ultracentrifugal analyses. Additional experience with this method has been gained, as it has been extensively applied to the study of the association of DNA repair enzymes, as described in more detail in the project report on physical biochemistry of macromolecules. The intensity approach has been extended by the incorporation of intensity methods into multiwavelength analyses for protein-DNA interactions, which has reduced the time for multiwavelength data acquisition by at least a third, and data processing and analysis time has been reduced by more than half. The second area of major development was the derivation of new numerical methods for the efficient solution of the Lamm equation, and the direct fitting of experimental sedimentation data using these numerical solutions of the Lamm equation. The new method exploits the moving frame of reference technique, which has been the basis for the well-known analytical approximate solutions of the Lamm equation and the g(s) technique, combining it with the finite element approach outlined by Claverie in 1976, and with modern global analysis approaches. This work has enabled the analysis of ideal non-interacting as well as self-associating species in a variety of novel experimental configurations. Very recently, in collaboration with Dr. Demeler, the direct Lamm analysis has been extended to the analysis of interference optical data which possess time-invariant noise components. This allows, for the first time, the hydrodynamic shape analysis of small molecules taking advantage of the enhanced sensitivity of the interference optical detection system of the analytical ultracentrifuge. In collaboration with the laboratory of Dr. A. Minton, the Lamm equation method has also been successfully employed in the Archibald analysis of the depletion of macromolecules in the vicinity of the meniscus at early times of a centrifuge experiment. This method can rapidly characterize the molar mass of proteins of limited thermal stability, by virtue of its substantially reduced experimental time. In collaboration with Dr. Howlett, we have started to extend the direct Lamm equation analysis approach further to enable the modeling of the sedimentation of particles with continuous molar mass distributions, such as lipid vesicles. Finally, instrumental developments in collaboration with Dr. Minton for the design of a fiber-optical absorbance scanning system for use in conjunction with post-centrifugal microfractionation have made substantial progress. This is a continuation of Intramural Research Project Z01-RR-10039-20 BEI.
| Lewis, Marc S; Reily, Michael M (2004) Estimation of weights for various methods of the fitting of equilibrium data from the analytical ultracentrifuge. Methods Enzymol 384:232-42 |
| Lebowitz, Jacob; Lewis, Marc S; Schuck, Peter (2002) Modern analytical ultracentrifugation in protein science: a tutorial review. Protein Sci 11:2067-79 |
| Dimitriadis, E K; Lewis, M S (2000) Optimal data analysis using transmitted light intensities in analytical ultracentrifuge. Methods Enzymol 321:121-36 |
| Kar, S R; Kingsbury, J S; Lewis, M S et al. (2000) Analysis of transport experiments using pseudo-absorbance data. Anal Biochem 285:135-42 |
