The overarching goal of the Synthetic Antigen Binder """"""""Sab Core"""""""" is to provide a broad range of powerful approaches and novel reagents to support the consortium's research objectives. The Sab Core is uniquely capable, based on the expertise and resources that have been developed under the auspices of the NIH Protein Structure Initiative (PSI), to generate in a high-throughput way synthetic antigen binders to an extensive range of targets including soluble proteins, protein complexes, membrane proteins and functional RNA. Thus, the infrastructure in place with extensive capability to efficiently generate high-quality affinity reagents will dramatically accelerate membrane protein research performed within the MPSD Consortium, as well as in the entire membrane protein research community. Our previous efforts had focused on generating Sabs for use as """"""""crystallization chaperones"""""""" (1). This endeavor has led to the crystallization and structure determination of several high-hanging fruit systems. Importantly, the Sab technology has a number of additional attributes that our team plans to exploit to investigate structure-dynamics-function relationships of membrane proteins in unique ways. Membrane proteins are dynamic machines that need to change their shape to perform their function. Therefore, it is critically important to functionally and structurally characterize major conformational states and determine how their populations are modulated during the course of action. Our Sabs are often exquisitely conformation-specific, making them powerful probes for studying protein conformation dynamics. They can be used to """"""""lock"""""""" a protein in a specific conformational state, which allows for unequivocal annotation of functional states (Fig. D3.1). Sabs can stabilize a specific conformational state to facilitate structural determination. They can also be engineered for use as affinity reagents to aid membrane protein purification as well as to stabilize membrane protein targets for storage. The importance of the last attribute cannot be overstated. Membrane proteins are inherently fragile. Since the proposed projects in the MPSD Consortium often involve shipment of samples between different locations, it is essential that they be delivered and stored in their native states. Further, Sabs can be used to attach spectroscopic probes to a specific location within a target with minimal modification to the target. The Sab Core produces three distinct classes of Sabs, in the forms of the antigen-binding fragment (Fab) of antibodies and other designer proteins that collectively fulfill diverse needs in membrane protein research. These Sabs are generated from high-performance phage-display libraries that are designed based on revolutionary concepts in protein engineering, and Sabs are produced in bacteria. It is our contention that, in the near future. Sabs will replace the traditional monoclonal antibody technology that is slow and expensive. The goals of the Sab Core are (i) to provide high-quality synthetic affinity reagents for membrane protein targets using state-of-the-art technologies, (ii) to accelerate structure determination by Sab-based chaperone-assisted crystallography and (iii) to develop novel applications of Sabs that will enable Core users to significantly elevate the level of mechanistic understanding of membrane protein functions. Sabs generated in this Core and ultimately the technology to produce Sabs will be made available to the broader scientific community.
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| Carnevale, Lauren N; Arango, Andres S; Arnold, William R et al. (2018) Endocannabinoid Virodhamine Is an Endogenous Inhibitor of Human Cardiovascular CYP2J2 Epoxygenase. Biochemistry 57:6489-6499 |
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