Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. The sensitivity of these measurements discerned that the 250's loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. Significance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability we provide direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop. Results Fully-automated and flexible pulse-labeling hydrogen/deuterium-exchange mass spectrometry with millisecond precisionOur goal was to quantify the structural switch in solution, along with other coincident dynamic changes in structure between activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of GlyP. To enable this, we developed a fully automated broadband bottom-up HDX-MS approach capable of accurate quantitative assessment of peptide stability. The newly developed ms2min system design ( Figure 1A) offers certain significant advances over the other designs previously employed. Notably, it allows fully-automated, software-selection of mixing times over six orders of magnitude (ms to hours) with 1 ms time resolution, flexible labeling temperature control (0 -25 C), quench temperature control (0 C), on-line connection for 'bottom-up' workflows, two-way communication for reciprocal control of labeling, washing, digestion, desalting and chromatography, automated digestion column wash injection, intercalated blank injections and sample list scheduling of multiple runs. Precision of D-labeling.To evaluate the measurement variability and labeling precision of the system we determined the repeatability of the quench-flow labeling method. On three separate days we measured the D uptake of 50, 70 and 150 replicates of Bradykinin and Leucine enkephalin at 100 ms mixing time. The short labeling period close to the limit of quantification, wa...
Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labeled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labeled protein (top-down) using soft gasphase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide-and ETD-levels to be combined; they can only be analyzed separately. Thus, we developed HDfleX, a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses, and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.
Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) experiments on protein structures can be performed at three levels: (1) by enzymatically digesting labelled proteins and analyzing the peptides (bottom-up), (2) by further fragmenting peptides following digestion (middle-down), and (3) by fragmenting the intact labelled protein (top-down), using soft gas-phase fragmentation methods, such as electron transfer dissociation (ETD). However, to the best of our knowledge, the software packages currently available for the analysis of HDX-MS data do not enable the peptide- and ETD-levels to be combined - they can only be analyzed separately. Thus, we developed HDfleX - a standalone application for the analysis of flexible high structural resolution of HDX-MS data, which allows data at any level of structural resolution (intact protein, peptide, fragment) to be merged. HDfleX features rapid experimental data fitting, robust statistical significance analyses and optional methods for theoretical intrinsic calculations and a novel empirical correction for comparison between solution conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.