SYNTHETIC BINDING PROTEINS

Cornell University, Ithaca NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Despite high-throughput methods, many important proteins remain unamenable to crystallization. Recently, an antibody-based strategy has evolved to counteract this. In this method, antibody antigen-binding fragments (Fabs) developed against the protein of interest are bound and the complex co-crystallized. This strategy stabilizes the target protein and provides non-target surface area for formation of crystal contacts, preventing aggregation and promoting lattice formation. Such chaperone assisted crystallography methods have proven successful in the crystallization of membrane proteins, and promise great utility for recalcitrant proteins generally. Some limitations of Fab usage for this purpose are low expression at high cost, redox sensitive disulfide linked structure, and large size which for reasons of target structure perturbation or packing may not be suitable in all instances. Furthermore, although synthetic technologies have emerged for the successful production of antibodies from combinatorial libraries, the theoretical size of these libraries often exceeds practical limits by many orders of magnitude resulting in incomplete sequence coverage and potential difficulty in obtaining binders for a given target. Our group has developed a much smaller antibody-mimic system using the Fibronectin Type III (FN3) scaffold for the generation of novel binding proteins. In particular, our group has recently established novel engineering technology to generate high-affinity binding proteins ("monobodies") employing severely restricted amino acid diversity in the target recognition region. The small size (~1/4 that of Fabs), the absence of disulfide bonds, and ease of production make the monobodies an attractive alternative to Fabs as crystallzation chaperones. Furthermore, small size coupled with low chemical diversity allows for complete sequence coverage in our combinatorial libraries. We have now determined the structures of 4 monobody-target complexes. Here we propose the use of APS resources to further explore the potential of monobody-assisted crystallography for protein structure determination. The data we obtain will be used to further optimize our combinatorial library design and inform future strategies for crystallization chaperone production.