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...

Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale. Typical HDX methods often cannot resolve the structural dynamics and stability in these cases. Numerous academic laboratories have demonstrated the considerable utility of acquiring HDX-MS data in the sub-second regimes. Here, we describe the development of a fully automated HDX-MS apparatus to resolve amide exchange on the millisecond timescale. Like conventional systems, this instrument boasts automated sample injection with software selection of labeling times, online flow mixing and quenching, while being fully integrated with a liquid chromatography− MS system for existing standard "bottom-up" workflows. HDX-MS's rapid exchange kinetics of several peptides demonstrate the repeatability, reproducibility, back-exchange, and mixing kinetics achieved with the system. Comparably, peptide coverage of 96.4% with 273 peptides was achieved, supporting the equivalence of the system to standard robotics. Additionally, time windows of 50 ms−300 s allowed full kinetic transitions to be observed for many amide groups; especially important are short time points (50−150 ms) for regions that are likely highly dynamic and solvent-exposed. We demonstrate that information on structural dynamics and stability can be measured for stretches of weakly stable polypeptides in small peptides and in local regions of a large enzyme, glycogen phosphorylase.

Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250′ and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen− deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.