# Membrane properties of the OHC system

> **NIH NIH R01** · YALE UNIVERSITY · 2021 · $167,075

## Abstract

Project Summary
In this proposal we seek to develop single molecule FRET in prestin. The method will allow us to assess the
function of the protein using optical measures. Single molecule FRET has been developed in other proteins to
aid in the understanding of their function. Presently, we do not have a functional assay of prestin with a single
molecule resolution.
The rationale for the experiments is based on structural data interpolated from the structure of several
members of the extended anion transporter family to which prestin belongs. Available structural data together
with modeling data suggest that members of this family have 14 transmembrane domains with an inverted
repeat 7+7 structure to each monomer. The functional units of these proteins appear to be dimers. Each
monomer has a gate and core domain. We model prestin to behave like these transporters using an elevator
mechanism to move from contracted to expanded states. An analogous movement in these family members
from inside open to outside open conformations result in a relative motion between two parts of the same
monomer. Thus, there is a movement of the gate domain (Tm domains 5-8 and 12-14) relative to the stable
core domain (Tm domains 1-4 and 9-11) containing the Tm anion binding site. Based on these models we will
insert several cysteine residues in the gate domain that will allow us to label a single dimer with two different
fluorophores with FRET activity. We have already generated a cysteine free protein that shows reduced but
measurable gating charge movement (NLC), confirming its functionality. The distance between the
fluorophores is modelled to vary by 30A0 that would give rise to a significant change in FRET efficiency.
Establishing a single molecule FRET assay will allow us to measure how physiological parameters affect the
function of the protein.

## Key facts

- **NIH application ID:** 10408895
- **Project number:** 3R01DC016318-05S1
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** JOSEPH R SANTOS-SACCHI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $167,075
- **Award type:** 3
- **Project period:** 2017-06-12 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10408895

## Citation

> US National Institutes of Health, RePORTER application 10408895, Membrane properties of the OHC system (3R01DC016318-05S1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10408895. Licensed CC0.

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