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 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 t be studied using traditional structural biology techniques. To overcome these barriers, a plan is proposed to develop and implement a set of novel methodologies and reagents based on phage display generated synthetic antibodies (sABs) that provide new means to study the molecular properties of transient states of membrane proteins at unprecedented detail. The concept is to use these sABs as customized crystallization chaperones or fiducial marks for single particle Cryo-EM applications. To advance these developments two powerful technology approaches will be combined. The first technology is a set of novel phage display sorting strategies that can endow the sABs with special properties providing investigators the tools to tackle complex structural and biological problems that were previously thought to be too daunting to even contemplate. The second technology advance is to perform these selections in lipid-filled nanodiscs that more faithfully recapitulate the native membrane environment. This environment will better stabilize desired conformational states induced by forces and as a consequence the selected sABs will capture and stabilize true dynamic intermediates that can be then studied by X-ray or Cryo-EM methods. To further increase success rates of these reagents as crystallization chaperones, antibody engineering will be employed to remodel the sAB scaffolds to effectively multiple the number of potential chaperones that can be derived from each sAB binder. To test and evaluate the methodologies, three classes of membrane ion-channels will be used as model systems. 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.

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)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Ainsztein, Alexandra M
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University of Chicago
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