Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Abstract

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...

HDfleX: Software for flexible high structural resolution of hydrogen/deuterium-exchange mass spectrometry data

“…Intrinsic rate calculation and protection factors. HDfleX also calculates the theoretical intrinsic rates for peptides 41 at the experimental pH and temperature. aSyn is incompatible with the Bai and Englander model for the calculation of intrinsic rates 27,44 as all the peptides exchanged faster than the predicted rates.…”

“…Calculation of intrinsic rates and protection factors. The intrinsic rates and protection factors were fitted and calculated as described previously 41 . Briefly, intrinsic uptake curves are generated, fitted into one-or two stretched exponentials.…”

Interconversion of unexpected thiol states affects stability, structure and dynamics in engineered antibody for site-specific conjugation

“…To investigate whether the thermodynamic and kinetic differences associated with the formation of an intramolecular disulfide bond were related to local changes in structure or dynamics, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments were conducted. This implementation of HDX-MS uses a novel fully-automated system capable measuring hydrogendeuterium exchange in the millisecond to minutes time scale [21] . Post labelling, online pepsin digestion was conducted and complete sequence coverage achieved in the resulting peptide map (Figure S. 9).…”

“…The Fc constructs were used as surrogates for C239i IgG since it has been well established that the antibody binding fragment (Fab) does not affect the stability of the CH2 and CH3 domains [19,20] . Chemical denaturation experiments, differential scanning calorimetry and millisecond HDX mass spectrometry [21] were used to probe the stability, dynamics and structures of the CH2 and CH3 domains in each of the enriched states. Together, these studies provide insight into the conditions under which different thiol states interconvert, their relative stabilities and the conformational changes that occur upon formation of an additional disulfide bond.…”

Structural Proteomics Methods to Interrogate the Conformations and Dynamics of Intrinsically Disordered Proteins

“…Kish et al developed a fully automated online flow mixing and quenching system, allowing reproducible HDX measurements between 50 ms and 5 min, with a 1 ms timescale resolution and temperature control (0–25°C) ( Kish et al, 2019 ). The authors used the setup to uncover novel mechanistic details regarding the structural dynamics involved in the allosteric regulation of GlyP.…”