# Chaperone-Assisted Structure Determination of Membrane Proteins

> **NIH NIH R01** · UNIVERSITY OF CHICAGO · 2021 · $364,500

## Abstract

Abstract:
 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.
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## Key facts

- **NIH application ID:** 10111523
- **Project number:** 5R01GM117372-06
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** ANTHONY A KOSSIAKOFF
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $364,500
- **Award type:** 5
- **Project period:** 2016-01-01 → 2023-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10111523

## Citation

> US National Institutes of Health, RePORTER application 10111523, Chaperone-Assisted Structure Determination of Membrane Proteins (5R01GM117372-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10111523. Licensed CC0.

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