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Updated: Jun 30, 2025
Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions
Published on: November 12, 2017
Time-Resolved Ultraviolet Photodissociation Mass Spectrometry Probes the Mutation-Induced Alterations in Protein
Pan Luo1,2,3,4, Zheyi Liu2,5, Can Lai2,3,5
1Institute of Advanced Science Facilities, Shenzhen 518107, China.
Abstract:
How mutations impact protein stability and structure dynamics is crucial for understanding the pathological process and rational drug design. Herein, we establish a time-resolved native mass spectrometry (TR-nMS) platform via a rapid-mixing capillary apparatus for monitoring the acid-initiated protein unfolding process. The molecular details in protein structure unfolding are further profiled by a 193 nm ultraviolet photodissociation (UVPD) analysis of the structure-informative photofragments. Compared with the wild-type dihydrofolate reductase (WT-DHFR), the M42T/H114R mutant (MT-DHFR) exhibits a significant stability decrease in TR-nMS characterization. UVPD comparisons of the unfolding intermediates and original DHFR forms indicate the special stabilization effect of cofactor NADPH on DHFR structure, and the M42T/H114R mutations lead to a significant decrease in NADPH-DHFR interactions, thus promoting the structure unfolding. Our study paves the way for probing the mutation-induced subtle changes in the stability and structure dynamics of drug targets.
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