Membrane proteins are complex molecular machines whose functions are governed by sets of programed conformational transitions. Attempts to establish the fundamental molecular mechanisms that link membrane protein structure and dynamics to functions they induce have been thwarted by a number of seemingly insurmountable technical barriers. Principal among these barriers is that the conformational transitions are too transient to be studied using traditional structural biology techniques. To overcome these barriers, we have developed and implemented a set of novel methodologies and reagents based on phage display generated synthetic antibodies (sABs). Customize phage display selection strategies enable generation of sABs endowed with special properties, for instance, conformation and regio-specificity. These reagents have been used to study the molecular properties of transient states of membrane proteins at unprecedented detail. While sABs have demonstrated efficacy as crystallization chaperones, their use in cryo-EM as powerful fiducial marks, adding 50 kDa to the particle and their ability to trap conformation states, is especially impactful in studies linking conformational transitions and function. This is particularly relevant for smaller membrane proteins (< 50 kDa), which include ion channels transporters and receptors. These constitute the largest class of biomedically relevant target systems, but are recalcitrant to crystallization and are far too small for cryo-EM analysis. Building on our current technology platform, we propose to design and deploy a set of higher-order sAB constructions that will serve to increase the size, rigidity and, in some cases, the symmetry of the target membrane protein. These sAB-based entities will be engineered to serve as prefabricated modules of assembly. They are targeted to specific epitopes that have been introduced into the membrane protein and thus, can be universally employed irrespective of the system they are applied to. The power of the approach is that these ?universal? sABs can be added to the molecule of interest in a ?plug and play? fashion allowing any investigator access to the powerful technology without requiring generating target specific sABs. To test and evaluate these novel sAB modules, we will use a set of high value small membrane proteins provided by investigators from our collaborator network. These systems have been recalcitrant to structural analysis using traditional approaches and thus, will provide a good measure of the performance of the chaperone-assisted structure determination technologies. An important byproduct is that these structures will provide valuable information about linkages between structure and dynamics that had been out of reach previously. !

Public Health Relevance

The Chaperone-Assisted Structure Determination (CSAD) pipeline provides novel classes of antibody-based reagents to further objectives in structural biology and membrane protein research. Membrane proteins are the principle drug targets in the pharmaceutical industry because they modulate myriad critical biological functions. The reagents produced are considerably more powerful than traditional antibodies because they can be targeted to specific sites on proteins and protein complexes and can be endowed with biological function. The CASD collaborates and services a large cohort of scientists whose interests span across multiple areas of high biomedical importance. These collaborations and the reagents that are produced are likely to lead to development of antibody-like leads that have significant therapeutic value.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Flicker, Paula F
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University of Chicago
Schools of Medicine
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