Fluorescence Anisotropy-based Macromolecule Crystallization Screening

DESCRIPTION (provided by applicant): Current practice is to set up trial crystallization screens and periodically review the results to see if a crystal or promising crystal-like precipitate has appeared a process that often takes weeks or months. Most out
comes are precipitated protein or clear drops, and the conditions that led to those results are dropped from further consideration. We propose an alternative screening approach, the self-association behavior of the target macromolecule as measured by fluor
escence anisotropy as a diagnostic for the likelihood of crystallization under the test conditions. Dilute solution properties are known to be a diagnostic for crystallization (George and Wilson, 1994; George et al., 1997; Wilson et al., 1993; Wilson et al
, 1996; Tessier et al., 2002; Tessier et al., 2003; Garcia et al., 2003a; Garcia et al., 2003b; Bloustine et al., 2003). Concentration vs. anisotropy data for a macromolecule-precipitant combination is proposed for determining the likelihood of that soluti
on producing crystals. Preliminary data indicate that this approach can find lead crystallization conditions from solutions that give clear drops or precipitate in screening assays. The applications of this instrument and methodology will be to rapidly c
onduct crystallization screens within 2-3 hrs, using a minimum amount of protein (= 0.7 mg at 10 mg/mL), with a higher probability of finding lead conditions. Higher success rates will greatly facilitate structure-based drug design, particularly for target
proteins that are difficult to obtain, and contribute to the understanding and treatment human disease. The Phase I proposal's objectives are to develop an instrument to make concentration vs. fluorescence anisotropy measurements, using = 100 fL of
macromolecule solution for a 96 condition screen, and then validate the performance with extensive testing. Long range this instrument will be the basis for a macromolecule crystallization business operated on a fee for service basis. Experience with a br
eadboard Phase 0 instrument has indicated where improvements can be made in the data collection and optics, and the initial Phase I work will be to assemble an improved instrument for making the anisotropy measurements. Subsequent testing will first be w
ith model proteins, obtained commercially or from a local collaborating structural genomics effort, using manually prepared solutions. For each model protein the concentration vs. anisotropy data obtained will be compared with crystallization screens set u
p in parallel, to define the signature curves indicating crystallization or potential crystallization outcomes and the extended data range over which crystallization conditions can be recovered. All anisotropy-derived leads will be tested with optimization
screens. Subsequent testing will be to challenge the methodology using previously uncrystallized soluble and membrane proteins from the same source. Projected subsequent Phase II efforts will be to reduce the amount of protein solution needed to = 10 nL,
to robotically prepare the assay solutions, and to automate data analysis with software developed on the basis of the data obtained. PUBLIC HEALTH RELEVANCE: Successful crystallization and X-ray data analysis provides important three-dimensional inf
ormation on the macromolecules structure-function relationship. Many proteins that are potential drug targets or key components in diseases are only available in trace quantities, or are difficult to obtain. This proposal is to develop a new approach to ma
cromolecule crystallization, using a minimum amount of protein, and giving data that can subsequently be analyzed to determine those conditions which will give crystals and those that can be brought to crystallization conditions, thus giving a higher succe
ss rate.