Proj 1: Improving macromolecule crystallization using symmetry-inducing GFP

Los Alamos Nat Secty-Los Alamos Nat Lab, Los Alamos NM

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Abstract

Summary/Abstract Improving macromolecular crystallization using lattice-promoting variants of GFP The bottleneck in structure determination by X-ray crystallography is crystallization, where -70% of purified proteins fail. A major reason proteins fail to crystallize is insufficient suitable lattice-forming contacts. The central hypothesis in this project is that the probability of obtaining crystals is increased every time a new form of a molecule with new potential crystal contacts is tested. We will develop rapid and general methods to generate numerous venations on a protein molecule by linking the protein to a series of oligomeric forms of green fluorescent protein (GFP). The use of GFP is a major advantage because it can be linked firmly to practically any protein by inserting a hairpin (two strands connected by a loop) from GFP into a loop of the target protein. The target protein containing this extra hairpin can bind tightly to a split version of GFP that is missing the hairpin. This means that we can create, in advance, a set of split-GFP oligomers with varying relationships between the monomers and varying surfaces^ The target protein or complex bearing the GFP hairpin inserted into one loop can be purified separately and then combined with each of the split-GFP modules, without any further genetic manipulation, protein expression, or purification of components, to yield a large number of different complexes that can be crystallized. Alternatively, the GFP modules themselves (rather than the hairpin) can be directly inserted into the target protein scaffold at permissive loops and turns. The GFP reagents and methods to use them will be validated on sets of model proteins. We hypothesize that the availability of these GFP scaffold reagents embodying different oligomeric forms will vastly expand the repertoire of possible lattice contacts, and substantially improve the crystallizability of proteins and protein complexes that may be otherwise difficult to crystallize. This work will lead to methods that will increase our understanding of human health and our ability to cure human illness by facilitating the structural determination of proteins involved in human disease.