# Membrane Protein Structure Using Evolutionary Couplings and Sparse NMR Data

> **NIH NIH R01** · RENSSELAER POLYTECHNIC INSTITUTE · 2020 · $519,776

## Abstract

PROJECT SUMMARY
Integral Membrane Proteins (IMPs) include many biomedically-important gate keepers, receptors,
transporters, homeostasis regulators, and potential drug discovery targets. Three-dimensional (3D)
structure determination of IMPs by X-ray crystallography, cryo-electron microscopy (cryo-EM), or
Nuclear Magnetic Resonance (NMR) methods remains a major challenge for structural biology.
While NMR can generally provide accurate 3D structures of small soluble proteins, structure
determination by solution NMR of IMPs, prepared in stabilizing membrane-mimicking environments
which generally require 2H,13C,15N-enrichment of the IMP, can be quite challenging. Evolutionary
couplings (ECs), evolutionarily-correlated mutations derived from multiple sequence alignments, can
also be used to provide information about native residue pair contacts, and to model the 3D
structures of IMPs. Combining EC and NMR data provides a powerful approach for overcoming
incompleteness of NMR NOESY data obtained for perdeuterated IMP samples, and the challenges in
identifying true native protein structure contacts from the phylogenetic EC analysis. In particular,
inter-helical contact information that is difficult to obtain for perdeuterated IMPs by NMR is well
represented in the sequence co-variance EC data. Our goals are to develop a robust, reproducible,
and fully automated EC-NMR platform suitable for accurate and reliable structure determination of
IMPs, particularly α-helical IMPs, and apply these methods for 3D structure analysis of biomedically-
important IMPs. EC-NMR will be further developed using β-barrel and α-helical IMPs of known
structure, and then applied to studies of IMPs of unknown structure selected from designated NIH
NIAID priority pathogenic bacteria. We will (i) further develop and apply the Single Protein Production
(SPP) method for producing isotope-enriched IMPs in E. coli, (ii) implement a micro-scale NMR
screening pipeline for IMP sample optimization, (iiii) rigorously and comprehensively address the
question of how EC and NOESY data quality and quantity correlate with the accuracy of EC-NMR
structures, (iv) design improved algorithms for structure determination of IMPs combining ECs and
NMR data, and (v) develop tools for validation of IMP structures determined by both conventional
NMR and EC-NMR methods. Advanced molecular modeling methods will be implemented to improve
accuracy of EC-NMR structures. ECs will also be combined with NMR data to identify and determine
structures of multiple “native states” of proteins. This study will expand the range of proteins that can
be studied by NMR, provide more accurate structural and dynamic information than can be obtained
with existing methods, and provide fundamental structural information needed for future antibiotic
drug development targeted to high-priority pathogens.

## Key facts

- **NIH application ID:** 9978825
- **Project number:** 5R01GM120574-05
- **Recipient organization:** RENSSELAER POLYTECHNIC INSTITUTE
- **Principal Investigator:** GAETANO T MONTELIONE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $519,776
- **Award type:** 5
- **Project period:** 2017-09-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9978825

## Citation

> US National Institutes of Health, RePORTER application 9978825, Membrane Protein Structure Using Evolutionary Couplings and Sparse NMR Data (5R01GM120574-05). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9978825. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*