Engineering Surface Epitopes to Improve Protein Crystallization
Lead Inventors: John Francis HuntProblem or Unmet Need:Attempts to prepare high-quality protein crystals suitable for structural analysis are expensive and time-consuming, yet often unsuccessful. The low success rate is rooted in the inability to assess the solubility of a protein or to predict its behavior in crystallization screens based on its amino acid sequence alone.Toward the goal of improving success rates, a metric to determine the probability of high-quality crystal formation would be useful in selecting targets for structural genomics or in determining structures to avoid the prospect of failing at the final step of the pipeline.This invention comprises a method of engineering protein surface epitopes for high-resolution X-ray crystallographic structure determination. The method is based on the use of a probability metric, which can predict to a high degree of accuracy whether or not high-quality protein crystals will form. The metric, in turn, is derived from analyses of 679 proteins that had passed through the Northeast Structural Genomics pipeline and were biologically well-behaved. Specifically, the metric is based on: examinations of those proteins for which detailed data was available across the entire production and crystallization process eliminations of external variables affecting protein crystallization such as solubility differences, expression levels and variations in experimental procedure evaluations of target success on the formation of sufficient-quality crystals to permit atomic resolution structure determinationSimple metrics based on sequence characteristics can predict which proteins are more likely to yield crystal structures; these results are statistically rigorous and were validated in an independent set of proteins. In addition, the metrics shed light on the critical role played by the availability of residues in the formation of crystals for high-resolution diffraction.
Metrics will reduce the time and cost of protein production and structure determination Significantly increase output in high-volume structural genomics initiatives Metrics can be used for target selection (sequence characteristics) and triage (biophysical characteristics), and could also yield novel target design strategies for challenging proteins.
The methods of the invention can be used to: better understand the mechanism of crystallization achieve rational target selection in high-throughput drug screening endeavors create mutants of targets in order to make them more easily crystallizableIn other words, the methods of this invention would allow to get a structure, which would then possibly show binding pocket locations. That could help in understanding of the active site and pocket structure, so that a drug, which binds to that location (and possibly competes with a known ligand), can eventually be identified.
This invention comprises a method of engineering protein surface epitopes for high-resolution X-ray crystallographic structure determination. The method is based on the use of a probability metric, which can predict to a high degree of accuracy whet...
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