SBIR-STTR Award

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 outcomes 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 fluorescence 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 solution 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 conduct 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 breadboard "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 with 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 up 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 information 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 macromolecule 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 success rate.

Public Health Relevance:
This Public Health Relevance is not available.

Thesaurus Terms:
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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 outcomes are precipitated protein or clear drops, and the conditions that led to those results are removed from further consideration. We are developing an alternative screening approach, the self-association behavior of the target macromolecule as measured by fluorescence 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 was originally proposed for determining the likelihood of that solution producing crystals, although based on Phase I results intensity vs. concentration data is also a strong indicator. Data acquired in Phase I shows that this approach can ""find"" lead crystallization conditions from solutions that give clear drops or precipitate in screening assays. The applications of the instrument and methodology to be developed will be to rapidly conduct crystallization screens within 2-24 hrs, using a minimum amount of protein ( 0.03 mg), 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 II proposal's objectives are to substantially improve the present instrument and methodology, progressing from the currently required 4.5 mg to 0.03 mg of protein/96 condition screen, and validate this method with extensive testing. This will be the basis for a macromolecule crystallization business operated on a fee-for-service basis. Experience with the Phase I instrument has indicated where improvements can be made in the plate set-up process, data collection optics, and electronics and the initial Phase II work will be to implement those improvements. Subsequent testing will first be with model proteins. For each model protein the concentration vs. anisotropy and intensity data obtained will be compared with crystallization screens set up 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. The model and test protein data will be used in the subsequent development of software for data analysis. Projected Phase III efforts include developing incomplete factorial screens that can make full use of the quantitative data obtained by this method. , ,

Public Health Relevance:
Successful crystallization and X-ray data analysis provides important three-dimensional information 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 continue development of a new approach to macromolecule crystallization, using a minimum amount of protein, and giving quantitative 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 success rate.

Thesaurus Terms:
Analysis, Data;Anisotropy;Appearance;Assay;Behavior;Bioassay;Biologic Assays;Biological Assay;Businesses;Collection;Computer Programs;Computer Software;Crystallization;Data;Data Analyses;Data Collection;Development;Development And Research;Diagnostic;Diffusion;Disease;Disorder;Drops;Drug Delivery;Drug Delivery Systems;Drug Design;Drug Targeting;Drug Targetings;Electronics;Fee-For-Service Plans;Fees For Service;Fluorescence;Fluorescence Anisotropy;Fluorescent Probes;Genes;Goals;Hgs;Hgs Protein;Hrs Protein;Instrumentation, Other;Label;Lead;Marketing;Measures;Membrane Proteins;Membrane-Associated Proteins;Method Loinc Axis 6;Methodology;Methods;Metric;Microfluidic;Microfluidics;Modeling;Modification;Optics;Outcome;Psi;Pb Element;Performance;Phase;Probability;Process;Property;Property, Loinc Axis 2;Protein Structure Initiative;Proteins;R & D;R&D;Radiation, X-Rays;Radiation, X-Rays, Gamma-Rays;Reliance;Research;Resolution;Roentgen Rays;Sbir;Sbirs (R43/44);Screening Result;Screening Procedure;Services;Small Business Innovation Research;Small Business Innovation Research Grant;Software;Solutions;Source;Structure;Structure-Activity Relationship;Surface Proteins;System;System, Loinc Axis 4;Testing;Time;Work;X-Radiation;X-Rays;Xrays;Base;Chemical Structure Function;Commercial Application;Computer Program/Software;Data Acquisition;Design;Designing;Develop Software;Developing Computer Software;Disease/Disorder;Experience;Gene Product;Gene Synthesis;Heavy Metal Pb;Heavy Metal Lead;Hepatocyte Growth Factor-Regulated Tyrosine Kinase Substrate;Hrs Gene Product;Human Disease;Improved;Instrument;Instrumentation;Macromolecule;New Approaches;Novel Approaches;Novel Strategies;Novel Strategy;Protein Function;Protocol Development;Public Health Relevance;Research And Development;Response;Screening;Screenings;Software Development;Structure Function Relationship;Success;Technological Innovation;Two-Photon