This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The field of protein design holds great promise for a wide range of applications: from design of therapeutics to tools for probing cell function to creating novel enzymes for biological and industrial processes. As a step towards the objective of generalizing protein-interaction design to a method for addressing problems in cell biology, we sought to design a novel protein-protein interface involving variants of an Ankyrin Repeat (AR) protein and a small a/b protein. We developed a general approach that incorporates a central hydrophobic cluster of sidechains at a protein-protein interface, which mimics naturally occurring hotspots at protein interfaces. We applied our general approach to create a set of de novo protein interfaces involving an AR protein and one of a collection of small a/b proteins. Of the fifteen designs experimentally tested, one was found to bind with a dissociation constant in the low mM affinity range. While in vitro evolution methods have allowed the successful engineering of interactions with higher affinities, this hotspot-based computational approach is a generally applicable method for the design of de novo protein-protein interfaces for normally non-interacting proteins. This methodology would have direct applications in the development of high affinity complexes suitable for protein affinity tags.While our current method requires sequence changes on each protein to optimize surface shape complementarity we aim to extend our current approach to alter the sequence of one of the partners to create high affinity complexes against naturally occurring targets. These new high affinity interactions could be used to supplant monoclonal antibodies for a variety of experiments including Western blots, affinity purification or even facilitate crystallization efforts.
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