Fluorescence Optics for the Analytical Ultracentrifuge

1) Purchase and commission XL-A upgrade to fluorescence optics. 2) Carry out determinations of protein/DNA dissociation constants. 3) Using tracer methodologies, understand better the effect of crowded environments upon macromolecular associations. 4) Use the equipment to derive experimental data which can be described by the new and emerging description of non-ideality described in the proposal.

The analytical ultracentrifuge can be used to determine both sedimentation coefficients and absolute molecular weights. It is a primary technique, requiring no calibration standards as is the case with gel filtration, and is thermodynamically rigorous; theoretically it is based directly on the second law of thermodynamics. New instrumentation in the early 1990's has lead to a renaissance in the technique with many new theoretical and methodological studies emerging every year. One limitation with the method, however, is that the optical system relies on either absorbance of light or refractive index increment (Rayleigh interference). This limits the minimum concentration to around 10 ug/ml. Practically, this means that dissociation constants can only be determined to around >200 nM. With fluorescence optics we will be able to probe concentrations down to 20-30 pM, and dissociation constants in the range >100 pM. In addition, fluorescently labelled macromolecules can be used to probe the effects of very high co-solute concentrations and therefore be a probe for how a particular interaction is affected by a crowded environment. Such non-ideal interactions can be analysed by an emerging methodology developed in the NCMH with our collaborators.