The bottleneck in structure determination by X-ray crystallography is crystallization, where -70% of purified proteins fail. The two major reasons proteins fail to enter the crystalline state are too few lattice contacts, and multiple conformations, often the result of missing partner proteins. This can be solved by the identification of missing partner proteins, the goal of one of the other projects in this proposal. An alternative approach, with the potential to address both problems is the subject of this proposal: the derivation of multimeric recombinant affinity scaffolds to protein crystallization targets. Antibody fragments are known to reduce protein conformations by fixing recognized proteins in one particular conformation, and Fab fragments have been used as crystallization partners, where contacts between the Fab domains substitute for the lack of contacts between target protein molecules. We propose to develop a suite of different recombinant binder libraries that can be easily selected against target proteins using phage display, and then reformatted into different oligomeric forms for use in crystallization studies. For example, it is known that the monomeric single chain Fv molecule can be transformed into a dimeric molecule by shortening the length of the linker connecting the heavy and light chain variable regions. This presents a novel strategy for binding and potentially rigidifying target proteins or protein complexes, while simultaneously introducing the favorable element of symmetry. For each target protein, several distinct scFvs selected for binding will be subsequently reformatted into a few different oligomeric arrangements. For any target protein or protein complex, these selected binder constructs will provide a suite of independent crystallization trials, each benefiting from the presence of a well-defined binding partner, as well as internal symmetry features conducive to crystallization. In addition to scFv libraries we will extend the strategy to create libraries based on stable fluorescent proteins that can be easily reformatted to dimers and tetramers, as well as using bacterial microcompartment shell proteins (e.g. CcmK4), which are relatively small and well-behaved, and form cyclic hexamers. We hypothesize that the availability of these three different binding scaffolds 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 hypothesis will be tested on protein complexes chosen from two target lists, articulated in Projects 1 and 3. The initial exploration of the use of these different oligomers will require significant protein purification. This will be facilitated by our choice of well-expressed stable scaffolds. Furthermore, as information is acquired on the utility of the different scaffolds and their oligomeric forms as crystallization chaperones, our production efforts will focus on those forms found to be most functional. This work will lead to methods that will increase our understanding of human health and our abilify to cure human illness by facilitating the structural determination of proteins involved in human disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-BCMB-A)
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Los Alamos National Lab
Los Alamos
United States
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Close, Devin W; Paul, Craig Don; Langan, Patricia S et al. (2014) Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure-guided surface engineering. Proteins :
Hart, Darren J; Waldo, Geoffrey S (2013) Library methods for structural biology of challenging proteins and their complexes. Curr Opin Struct Biol 23:403-8