Wednesday, April 2, 2025 4/2/2025

Linking the conformational landscape to enzymatic function through functional site distant mutations

Award Number: R01GM144349
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

Group Awards By:

View Award Description

Linking the conformational landscape to enzymatic function through functional site distant mutations - An ongoing debate concerns the role conformational motions, often termed dynamics, play in biomolceular funtion. For enzymes, it so happens that the timescales for large-scale domain motions are similar to the apparent $$). catalytic rate (𝑘! # This observation is where the major point of contention has developed: do $$. In this context, the proposed studies conformational motions directly impact the true rate of catalysis (𝑘! #) or 𝑘! # will further explore how the modulation of the conformational landscape can indeed fine-tune $$ without 𝑘! # impacting 𝑘! # and the ground state structure. The genesis of this proposal arises from our work with human guanylate kinase (hGMPK), a potential therapeutic target for treating cancer and perhaps even SARS-CoV-2, which motivated us to solve the first structure of hGMPK with nuclear magnetic resonance (NMR) spectroscopy (PDB: 6NUI). While solving the hGMPK structure, we expressed a series of seven functional site distant (FSD), $$ when compared to the wild- non-synonymous single nucleotide variants (nsSNVs) of hGMPK that enhance 𝑘! # type (wt). Intriguingly, the 2D [1H,15N]-HSQC NMR spectra of the wt hGMPK and its nsSNVs suggest that the 𝑎𝑝𝑝) for GMP binding to FSD mutations minimally impact hGMPK’s backbone fold, yet the apparent off-rates (𝑘𝑜𝑓𝑓 wt and the FSD mutant V91M differ by ~3000 s-1. We hypothesize that hGMPK’s activity can be modulated with FSD mutants by reshaping the conformational landscape. Utilizing NMR spectroscopy and isothermal calorimetry, we will test this hypothesis in the following two Specific Aims. In Aim 1, we will quantify the impact of the FSD mutations on the conformational landscape from kinetic and thermodynamic perspectives. The results from this Aim will provide a comprehensive picture as to where within the hGMPK catalytic and binding schemes the FSD mutations have the largest impact on function. For Aim 2, we will deconvolute the contribution transient structures within the conformational landscape play in enzymatic catalysis through experimentally driven ensemble generation. Our protocol will select hGMPK structures from unbiased molecular dynamics (MD) simulations based on residual dipolar couplings and cross-correlated relaxation rates measured with NMR. The ensembles will aid in the identification of the functionally important transient conformations and an assessment of the impact the FSD mutations have on backbone dihedral correlated motions. To our knowledge, this proposal provides the first examples 1.) of experimentally driven, ensemble generation for an enzyme spanning physiologically relevant timescales and 2.) of determining thermodynamic and kinetic parameters with ligand binding to the same exact site on a series of enzyme variants. The impact of this proposal is the direct linkage of the conformational landscape to enzymatic function. Immediate applications for these results include drug discovery, where targeting structures within the conformational landscape rather than the ground state structure will lead to better outcomes, and biomolecular design, where FSD mutations can be implemented to adjust function through manipulation of the conformational landscape.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2025 ( Subtotal = $291,736 )
2025	2025	UNIVERSITY OF LOUISVILLE	2301 S 3RD ST	LOUISVILLE	KY	40208	JEFFERSON	USA	Biomedical Research and Research Training	000	4	12/16/2024	NON-COMPETING CONTINUATION	$291,736
														Subtotal = $291,736

Issue Date FY: 2024 ( Subtotal = $324,282 )
2024	2024	UNIVERSITY OF LOUISVILLE	2301 S 3RD ST	LOUISVILLE	KY	40208	JEFFERSON	USA	Biomedical Research and Research Training	001	3	6/18/2024	NON-COMPETING CONTINUATION	$32,428
2024	2024	UNIVERSITY OF LOUISVILLE	2301 S 3RD ST	LOUISVILLE	KY	40208	JEFFERSON	USA	Biomedical Research and Research Training	000	3	12/12/2023	NON-COMPETING CONTINUATION	$291,854
														Subtotal = $324,282

Issue Date FY: 2023 ( Subtotal = $324,409 )
2023	2023	UNIVERSITY OF LOUISVILLE	2301 S 3RD ST	LOUISVILLE	KY	40208	JEFFERSON	USA	Biomedical Research and Research Training	000	2	12/6/2022	NON-COMPETING CONTINUATION	$291,968
2023	2023	UNIVERSITY OF LOUISVILLE	2301 S 3RD ST	LOUISVILLE	KY	40208	JEFFERSON	USA	Biomedical Research and Research Training	001	2	2/20/2023	NON-COMPETING CONTINUATION	$32,441
														Subtotal = $324,409

Issue Date FY: 2022 ( Subtotal = $324,025 )
2022	2022	UNIVERSITY OF LOUISVILLE	2301 S 3RD ST	LOUISVILLE	KY	40208	JEFFERSON	USA	Biomedical Research and Research Training	000	1	12/21/2021	NEW	$324,025
														Subtotal = $324,025

Grand Total All Awards = $1,264,452

Top

All Categories

About

Search

Reports

Data Submission

Award Information

Linking the conformational landscape to enzymatic function through functional site distant mutations

Award Number: R01GM144349

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

Federal Websites

Department of Health & Human Services

HHS Operating Divisions

HHS Staff Divisions

Download A Document Viewer