Peter Liuni

Conformer Selection and Intensified Dynamics During Catalytic Turnover in Chymotrypsin

During catalytic turnover, enzymes undergo thermally driven conformational fluctuations (dynamics) that are directly linked to catalytic efficiency. In broad terms, this link exists because enzymes must sample specific dynamic modes in order to access high-energy structures along the catalytic reaction coordinate. Mass spectrometry-based approaches for probing enzyme dynamics are developing rapidly, but have been limited thus far by the application of H/D exchange (HDX) under steady-state conditions, which results in averaging of the data over multiple catalytic states. 6] Models that attempt to define the specific nature of the enzyme dynamics/activity relationship are therefore drawn overwhelmingly from Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion NMR experiments on “active” enzymes. 7,8] This approach is extremely powerful, but is confined to a small set of highly reversible reactions in order to circumvent the issue of substrate depletion during the experiment. 3, 9–12] From the limited set of “CPMG accessible” reactions, two models for catalysis-linked dynamics have been formulated. The “induced fit” model is characterized by a substrate-free (resting) state that samples a different set of dynamic modes compared to the substrate-bound, catalytically active state. This would imply that the dynamics observed in the resting state should be substantially different from those observed during catalysis. A number of studies have reported evidence supporting this model. 14] In the “conformer selection” model, the conformational space sampled by the enzyme is independent of catalysis (i.e., the resting and active-state conformational dynamics are identical). Productive enzyme– substrate interactions occur when incoming substrate “selects” the appropriate conformer for binding. Conformer selection is supported by a substantial number of studies showing no difference in dynamics between free and substrate-bound enzyme. 3, 5,9–11] “Hybrid” models have also been proposed in which substrate binding occurs through conformer selection followed by “induced fit-like” substratedirected conformational sampling during catalysis. These models provide crucial insights into virtually all aspects of enzyme function including substrate binding, specificity, allostery and rate-limiting catalytic processes. However, their formulation from a relatively small pool of similar enzyme systems suggests that they may describe only a fraction of possible catalysis-linked dynamic modes. In this work, we probe conformational dynamics in an active, “CPMG inaccessible” enzyme system using an alternative approach that combines time-resolved electrospray mass spectrometry (TRESI-MS) and sub-second H/D exchange (HDX) labeling to monitor dynamics in the pre-steady state. By this approach, catalytic processes are detected as time-dependent intensity changes in mass-tocharge (m/z) peaks corresponding to the accumulation and/or depletion of enzyme intermediates. For each species that becomes populated during the measurement, dynamics are probed simultaneously, by the rate and magnitude of deuterium uptake. In contrast to CPMG NMR spectroscopy, these measurements are not “site specific”, however, they represent a straightforward and broadly applicable method for characterizing dynamics in active enzyme systems. A schematic illustration of the experimental setup is provided in Figure 1.

Understanding and optimizing electrospray ionization techniques for proteomic analysis

Electrospray ionization (ESI) mass spectrometry is a powerful and versatile tool for proteomic analysis. By understanding how proteins and peptides behave during ESI, it is possible to predict source conditions that will maximize ionization efficiency, ultimately leading to lower detection limits for protein identification and more accurate quantitation. In this article, we provide an overview of a variety of electrospray-based ionization methods, including nanospray, liquid chromatography and capillary electrophoresis-coupled sources, and how they are optimized for proteomic samples. We will touch upon analyte characteristics, solvent/eluent conditions as well as optimization of ESI for top-down, bottom-up and quantitative experiments.

Conformational Dynamics and Allostery in Pyruvate Kinase.

Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the “domain rotation model” suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the “rigid body reorientation model” proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.

CHAPTER 8:Studying Enzyme Mechanisms Using Mass Spectrometry, Part 2: Applications

Enzymes are involved in nearly all biological processes, catalyzing reactions with exceptional selectivity and efficiency. Knowledge of reaction mechanisms, whereby a substrate is converted to product through one or more intermediates, is essential for unravelling an enzyme’s role in metabolism. The most used analytical methods for the study of enzyme function include UV–visible fluorescence spectroscopy, X-ray crystallography, two-dimensional nuclear magnetic resonance, and isothermal calorimetry. However, these methods have several limitations that need to be overcome for the detection of transiently populated intermediate states. Recently, mass spectrometry has proven to be an efficient and sensitive method for studying the structural changes occurring at both the protein and substrate level during enzymatic turnover. This chapter will focus on how mass spectrometry has been successfully applied to several unique systems for the elucidation of enzyme reaction mechanisms, kinetic isotope effects, binding constants and catalysis-linked dynamics.

CHAPTER 7:Studying Enzyme Mechanisms Using Mass Spectrometry, Part 1: Introduction

Enzymes are involved in nearly all biological processes, catalyzing reactions with exceptional selectivity and efficiency. Knowledge of reaction mechanisms, whereby a substrate is converted to product through one or more intermediates, is essential for unravelling an enzyme’s role in metabolism. The most used analytical methods for the study of enzyme function include UV–visible fluorescence spectroscopy, X-ray crystallography, two-dimensional nuclear magnetic resonance, and isothermal calorimetry. However, these methods have several limitations that need to be overcome for the detection of transiently populated intermediate states. Recently, mass spectrometry has proven to be an efficient and sensitive method for studying the structural changes occurring at both the protein and substrate level during enzymatic turnover. Chapter 8 will focus on how mass spectrometry has been successfully applied to several unique systems for the elucidation of enzyme reaction mechanisms, kinetic isotope effects, binding constants and catalysis-linked dynamics.

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.