# Membrane Protein Structure Using Evolutionary Couplings and Sparse NMR Data

> **NIH NIH R01** · RENSSELAER POLYTECHNIC INSTITUTE · 2020 · $249,820

## Abstract

PROJECT SUMMARY
This Supplement to parent grant 1R01 GM120574 requests funds to purchase a Helium Recovery System.
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, 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 with 2H,13C,15N-isotope
enrichment in stabilizing membrane-mimicking environments, can be quite challenging. Evolutionary couplings
(ECs), evolution-based contact predictions derived using bioinformatics methods from multiple sequence
alignments, can also provide useful information for modeling the 3D structures of IMPs. By combining EC and
NMR data, we can overcome the incompleteness of NMR NOESY data obtained for perdeuterated IMP
samples, and address challenges in identifying true native protein structure contacts (true positives) from the
phylogenetic EC analysis. In particular, inter-helical contact information that is difficult to obtain for
perdeuterated IMPs by NMR is generally well represented in the sequence co-variance EC data. The EC-NMR
method will be developed using b-barrel and a-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)
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, (iii) design improved
algorithms for structure determination of IMPs combining ECs and NMR data, and (iv) develop standards and
tools for validation of IMP structures determined by EC-NMR methods. ECs will also be combined with NMR
data to identify and determine structures of multiple “native states” of proteins. Liquid Helium (LHe) is
essential for operation of the high field 500 MHZ, 600 MHz, and 800 MHz NMR systems used in this project.
LHe supplies have become limited, and our delivery quotas from the vendor, Air Gas, Inc, are insufficient to
maintain our superconducting NMR and MRI magnets. LHe prices have also increased 3-fold over the past few
years, and the quality of LHe has been inconsistent, which can damage the cryostats. Several NIGMS- (and
other NIH- and NSF-) funded projects depend on superconducting magnets maintained in the RPI Magnetic
Resonance Core Facility. These NMR and MRI systems represent a significant investment by RPI and NIH, and
are at risk for shutdown due to LHe cost and accessibility. Hence, it is critical that we obtain a Helium Recovery
System. Multiple systems have been evaluated. Both installation and long-term maintenance plans are well-
developed. The pr...

## Key facts

- **NIH application ID:** 10134530
- **Project number:** 3R01GM120574-05S1
- **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:** $249,820
- **Award type:** 3
- **Project period:** 2017-09-01 → 2022-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10134530

## Citation

> US National Institutes of Health, RePORTER application 10134530, Membrane Protein Structure Using Evolutionary Couplings and Sparse NMR Data (3R01GM120574-05S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10134530. Licensed CC0.

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