Work

Studying Protein-Ligand Interactions with In-droplet Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics Simulations

Year: 2026

Type: preprint

Abstract: Native mass spectrometry (nMS) has been widely used for the characterization of biomolecules and their interactions with ligands under near-physiological conditions, yet connecting the change in conformational dynamics of proteins upon ligand binding remains challenging. In this work, the integration of capillary vibrating sharp-edge spray ionization based-nMS, in-droplet hydrogen deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations provides a platform to characterize ligand binding and ligand-induced conformational changes of bovine carbonic anhydrase II (BCAII) upon interaction with its inhibitor, acetazolamide (AZM). cVSSI-based-nMS preserves the protein native-like state and facilitates the direct identification and determination of the apparent binding affinity of the BCAII-AZM complex. In-droplet HDX-MS measurements show a reduction in deuterium incorporation upon AZM binding suggesting ligand-induced structural stabilization of the holo BCAII. MD simulations of the apo and holo protein revealed ligand-induced suppression in backbone fluctuations and enhanced structural rigidity. A new model that uses protection factors (PF) and HDX scoring derived from MD trajectories that is based on solvent accessible surface area (SASA) and hydrogen bonding propensity is further refined. The model reflects experimentally observed trends in deuterium incorporation and offers atomic-level insight into structural stabilization of BCAII upon AZM binding. The combination of the cVSSI-based nMS, in-droplet HDX-MS and MD simulations provides a framework for identification and quantification of protein-ligand interactions and elucidation of ligand-induced changes in conformational dynamics of the protein.

Source: ChemRxiv

DOI: 10.26434/chemrxiv.15008038/v1

Authors: Sohag Ahmed, Mst Nigar Sultana, Md Reaz Uddin, Sultan Mahmud, Mohammad A. Rahman, Peng Li, Stephen J. Valentine

Institutions: West Virginia University, University of Louisville, Kennesaw State University, Indiana University Bloomington, Indiana University, Lehigh University

Language: English

Topic: Mass Spectrometry Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

Open Access status: green

Funder: National Institute of General Medical Sciences

Award: Developing an integrated system for detailing the conformational flexibility, heterogeneity, and dynamics of functional oligonucleotides

OpenAlex ID: W7204815523

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