Hydrogen-deuterium Exchange Kinetics Research Articles

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Alkyne-Tagged Raman Probes for Local Environmental Sensing by Hydrogen-Deuterium Exchange.

Alkyne-tagged Raman probes have shown high promise for noninvasive and sensitive visualization of small biomolecules to understand their functional roles in live cells. However, the potential for alkynes to sense cellular environments that goes beyond imaging remains to be further explored. Here, we report a general strategy for Raman imaging-based local environment sensing by hydrogen-deuterium exchange (HDX) of terminal alkynes (termed alkyne-HDX). We first demonstrate, in multiple Raman probes, that deuterations of the alkynyl hydrogens lead to remarkable shifts of alkyne Raman peaks for about 130 cm-1, providing resolvable signals suited for imaging-based analysis with high specificity. Both our analytical derivation and experimental characterizations subsequently establish that HDX kinetics are linearly proportional to both alkyne pKas and environmental pDs. After validating the quantitative nature of this strategy, we apply alkyne-HDX to sensing local chemical and cellular environments. We establish that alkyne-HDX exhibits high sensitivity to various DNA structures and demonstrates the capacity to detect DNA structural changes in situ from UV-induced damage. We further show that this strategy is also applicable to resolve subtle pD variations in live cells. Altogether, our work lays the foundation for utilizing alkyne-HDX strategy to quantitatively sense the local environments for a broad spectrum of applications in complex biological systems.

Reactivity of internal vs. external Brønsted acid sites in nanosponge MFI: H/D exchange kinetic study

The strength of Brønsted acid sites (BAS) affects the properties of 2D and hierarchical zeolites, but the relative contribution of internal and external BAS remains unknown. Accordingly, this study aims to assess the acidity of external and internal BAS in nanosponge-like MFI zeolites by comparatively analyzing hydrogen–deuterium exchange kinetics between zeolitic deuteroxyl groups and C 2 H 6 molecules monitored by in-situ FTIR spectroscopy. For this purpose, (i) a sample pre-treatment procedure was specifically developed to deuterate only internal or only external acid sites using 2,6-di-tert-butylpyridine (DTBP) as a masking agent and (ii) DFT modeling of surface BAS was performed. Theoretical models of the thin MFI layer revealed that the external surface of MFI crystals contains three types of BAS: (i) BAS positioned in 5-membered rings, either shielded by silanol nests, rendering the site inaccessible for DTBP or yielding a very low adsorption energy for ethane, (ii) BAS pointing into the pores, due to the presence of aluminol, which hinders DTBP accessibility, or the BAS undergoes transformation to a three-coordinate aluminium site, and (iii) BAS accessible to both DTBP and ethane. The results from our kinetics measurements showed that H/D exchange at external BAS of nanosponge MFI zeolites is faster than at internal BAS (rate constants at 425 °C: 3.8 10 −3 vs. 2.4 10 −3 s −1 for external and internal BAS, respectively), but this cannot be attributed to the effect of diffusion. Therefore, the differences in exchange kinetics between external and internal BAS are given by mutual interplay of subtle differences in the corresponding activation barriers (113 vs. 117 kJ/mol for external and internal BAS, respectively) and pre-exponential terms (1.09 10 6 vs. 1.45 10 6 s −1 for external and internal BAS, respectively). • Treatment procedure to discriminate internal and external Brønsted acid sites proposed. • Three types of external Brønsted acid sites was described based on DFT modeling. • Strength of internal and external Brønsted acid sites is the same.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, andexert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

Characterization and Management of Noise in HDX-MS Data Modeling.

Quantification of hydrogen deuterium exchange (HDX) kinetics can provide information on the stability of individual amino acids in proteins by finding the degree to which the local backbone environment corresponds to that of a random coil. When characterized by mass spectrometry, extraction of HDX kinetics is not possible because different residue exchange rates become merged depending on the peptides that are formed during proteolytic digestion. We have recently developed an advanced programming tool called HDXmodeller, which enables the exchange rates of individual amino acids to be understood by optimization of low-resolution HDX-mass spectrometry (MS) data. HDXmodeller is also uniquely able to appraise each optimization and quantify the accuracy of modeled exchange rates ab initio using a novel autovalidation method based on a covariance matrix. Here, we address the noise-handling capabilities of HDXmodeller and demonstrate the effectiveness of the algorithm on self-inconsistent datasets. Reference intervals for experimental HDX-MS data are also derived, and this information is presented in an updated online workflow for HDXmodeller, allowing users to evaluate the consistency of their data. The development of a modified version of HDXmodeller is also discussed with enhanced noise-handling capability brought about through loss function optimization. Changes in optimizer accuracy with different loss functions are also demonstrated along with the effectiveness of HDXmodeller to select the most effective optimizer for different data using currently embedded autovalidation criteria.

Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry.

Protein digestion is a key challenge in mass spectrometry (MS)-based structural proteomics. Although using hydrogen-deuterium exchange kinetics with MS (HDX-MS) to interrogate the high-order structure of proteins is now established, it can be challenging for β-barrel proteins, which are important in cellular transport. These proteins contain a continuous chain of H-bonds that impart stability, causing difficulty in digestion for bottom-up measurements. To overcome this impediment, we tested organic solvents as denaturants during on-line pepsin digestion of soluble β-barrel proteins. We selected green fluorescent protein (GFP), siderocalin (Scn), and retinol-binding protein 4 (RBP4) as model proteins and screened six different polar-aprotic and polar-protic solvent combinations to disrupt the H-bonds and hydrophobic interactions holding together the β-sheets. The use of organic solvents improves digestion, generating more peptides from the rigid β-barrel regions, without compromising the ability to predict the retinol binding site on RBP4 when adopting this proteolysis with HDX.

Development of Software for the In-Depth Analysis of Protein Dynamics as Determined by MALDI Mass Spectrometry-Based Hydrogen/Deuterium Exchange.

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated software. Currently available software programs mainly calculate average mass shifts, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, HDX reaction samples are typically composed of peptides that contain various numbers of deuterium atoms, which also hinders the rapid and comprehensive analysis of protein dynamics. We report here on the development of a software program “Scipas DX” that can be used to automatically analyze the hydrogen–deuterium isotopic distribution in peaks in HDX spectra and calculate the average number of atoms exchanged, the average deuteration ratio, the abundance ratio for exchanged atoms, and their fitted spectra with a high degree of accuracy within a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicate that the local structure at residues 83–106 and 107–117 are in a slow equilibrium, suggesting that these regions adopt multiple conformations that are involved in the stability and in switching between the active and inactive forms. Furthermore, precise HDX kinetics of the average deuteration ratio both confirmed the known induced conformations of two regions (residues 46–75 and 131–165) that are responsible for ligand binding and verified the novel structural dynamics of residues 107–117 and 166–196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analyses of protein dynamics.

An Integrated Approach for Determining a Protein-Protein Binding Interface in Solution and an Evaluation of Hydrogen-Deuterium Exchange Kinetics for Adjudicating Candidate Docking Models.

We describe an integrated approach of using hydrogen-deuterium exchange mass spectrometry (HDX-MS), chemical cross-linking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics reports widespread protection on IL-7Rα but shows no differential evidence of binding-induced protection or remote conformational change. Cross-linking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular cross-link-derived restraints are sufficient to generate a high-confidence model with root-mean-square distance (rmsd) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of interprotein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.

Arabinose Alters Both Local and Distal H-D Exchange Rates in the Escherichia coli AraC Transcriptional Regulator.

In the absence of arabinose, the dimeric Escherichia coli regulatory protein of the l-arabinose operon, AraC, represses expression by looping the DNA between distant half-sites. Binding of arabinose to the dimerization domains forces AraC to preferentially bind two adjacent DNA half-sites, which stimulates RNA polymerase transcription of the araBAD catabolism genes. Prior genetic and biochemical studies hypothesized that arabinose allosterically induces a helix-coil transition of a linker between the dimerization and DNA binding domains that switches the AraC conformation to an inducing state [Brown, M. J., and Schleif, R. F. (2019) Biochemistry, preceding paper in this issue (DOI: 10.1021/acs.biochem.9b00234)]. To test this hypothesis, hydrogen-deuterium exchange mass spectrometry was utilized to identify structural regions involved in the conformational activation of AraC by arabinose. Comparison of the hydrogen-deuterium exchange kinetics of individual dimeric dimerization domains and the full-length dimeric AraC protein in the presence and absence of arabinose reveals a prominent arabinose-induced destabilization of the amide hydrogen-bonded structure of linker residues (I167 and N168). This destabilization is demonstrated to result from an increased probability to form a helix capping motif at the C-terminal end of the dimerizing α-helix of the dimerization domain that preceeds the interdomain linker. These conformational changes could allow for quaternary repositioning of the DNA binding domains required for induction of the araBAD promoter through rotation of peptide backbone dihedral angles of just a couple of residues. Subtle changes in exchange rates are also visible around the arabinose binding pocket and in the DNA binding domain.

Evidence for a Partially Stalled γ Rotor in F1-ATPase from Hydrogen-Deuterium Exchange Experiments and Molecular Dynamics Simulations.

F1-ATPase uses ATP hydrolysis to drive rotation of the γ subunit. The γ C-terminal helix constitutes the rotor tip that is seated in an apical bearing formed by α3β3. It remains uncertain to what extent the γ conformation during rotation differs from that seen in rigid crystal structures. Existing models assume that the entire γ subunit participates in every rotation. Here we interrogated E.coli F1-ATPase by hydrogen-deuterium exchange (HDX) mass spectrometry. Rotation of γ caused greatly enhanced deuteration in the γ C-terminal helix. The HDX kinetics implied that most F1 complexes operate with an intact rotor at any given time, but that the rotor tip is prone to occasional unfolding. A molecular dynamics (MD) strategy was developed to model the off-axis forces acting on γ. MD runs showed stalling of the rotor tip and unfolding of the γ C-terminal helix. MD-predicted H-bond opening events coincided with experimental HDX patterns. Our data suggest that in vitro operation of F1-ATPase is associated with significant rotational resistance in the apical bearing. These conditions cause the γ C-terminal helix to get "stuck" (and unfold) sporadically while the remainder of γ continues to rotate. This scenario contrasts the traditional "greasy bearing" model that envisions smooth rotation of the γ C-terminal helix. The fragility of the apical rotor tip in F1-ATPase is attributed to the absence of a c10 ring that stabilizes the rotation axis in intact FoF1. Overall, the MD/HDX strategy introduced here appears well suited for interrogating the inner workings of molecular motors.

Ion Mobility Spectrometry-Mass Spectrometry Coupled with Gas-Phase Hydrogen/Deuterium Exchange for Metabolomics Analyses.

Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract. Graphical Abstract ᅟ.

Peptide-Level Interactions between Proteins and Small-Molecule Drug Candidates by Two Hydrogen−Deuterium Exchange MS-Based Methods: The Example of Apolipoprotein E3

We describe a platform utilizing two methods based on hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to characterize interactions between a protein and a small-molecule ligand. The model system is apolipoprotein E3 (apoE3) and a small-molecule drug candidate. We extended PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) to the regional level by incorporating enzymatic digestion to acquire binding information for peptides. In a single experiment, we not only identified putative binding sites, but also obtained affinities of 6.0, 6.8, and 10.6 μM for the three different regions, giving an overall binding affinity of 7.4 μM. These values agree well with literature values determined by accepted methods. Unlike those methods, PLIMSTEX provides site-specific binding information. The second approach, modified SUPREX (stability of unpurified proteins from rates of H/D exchange) coupled with electrospray ionization (ESI), allowed us to obtain detailed understanding about apoE unfolding and its changes upon ligand binding. Three binding regions, along with an additional site, which may be important for lipid binding, show increased stability (less unfolding) upon ligand binding. By employing a single parameter, ΔC1/2%, we compared relative changes of denaturation between peptides. This integrated platform provides information orthogonal to commonly used HDX kinetics experiments, providing a general and novel approach for studying protein-ligand interactions.

Hydrogen–Deuterium exchange kinetics in β-lactoglobulin (−)-epicatechin complexes studied by FTIR spectroscopy

Hydrogen–Deuterium exchange kinetics of β-lactoglobulin and β-lactoglobulin (−)-epicatechin solutions has been investigated through the analysis of the amide I absorption band at 1650cm−1 in the FTIR spectrum. H–D substitution in NH amides and residues of the protein results in a slight red-shift and in intensity changes of the amide I components: either these effects have been inspected in the framework of the Principal Components Analysis methods. The present analysis allowed to unveil three H–D kinetics at the timescale of the oligomeric fluctuations of the protein. A fast mechanism (lifetime from 5 to 10min) can be ascribed to the dynamics of protein oligomers and aggregates at the scale of the quaternary structure variations, and it is not observed in the complexes β-lactoglobulin (−)-epicatechin. The other slowest kinetics, whose lifetimes are in the range 1–10h, are here associated to dynamics of high-molecular weight complexes that hamper the proton exchange. The role of (−)-epicatechin as an enhancer of the formation of stable high-molecular weight aggregates from β-lactoglobulin is also discussed by comparison of the lifetimes at different protein concentrations.

Screen-printed digital microfluidics combined with surface acoustic wave nebulization for hydrogen-deuterium exchange measurements

An inexpensive digital microfluidic (DMF) chip was fabricated by screen-printing electrodes on a sheet of polyimide. This device was manually integrated with surface acoustic wave nebulization (SAWN) MS to conduct hydrogen/deuterium exchange (HDX) of peptides. The HDX experiment was performed by DMF mixing of one aqueous droplet of angiotensin II with a second containing various concentrations of D2O. Subsequently, the degree of HDX was measured immediately by SAWN-MS. As expected for a small peptide, the isotopically resolved mass spectrum for angiotensin revealed that maximum deuterium exchange was achieved using 50% D2O. Additionally, using SAWN-MS alone, the global HDX kinetics of ubiquitin were found to be similar to published NMR data and back exchange rates for the uncooled apparatus using high inlet capillary temperatures was less than 6%.

Protein-like proton exchange in a synthetic host cavity

The mechanism of proton exchange in a metal-ligand enzyme active site mimic (compound 1) is described through amide hydrogen-deuterium exchange kinetics. The type and ratio of cationic guest to host in solution affect the rate of isotope exchange, suggesting that the rate of exchange is driven by a host whose cavity is occupied by water. Rate constants for acid-, base-, and water-mediated proton exchange vary by orders of magnitude depending on the guest, and differ by up to 200 million-fold relative to an alanine polypeptide. These results suggest that the unusual microenvironment of the cavity of 1 can dramatically alter the reactivity of associated water by magnitudes comparable to that of enzymes.

Gas-Phase Hydrogen-Deuterium Exchange Labeling of Select Peptide Ion Conformer Types: a Per-Residue Kinetics Analysis.

The per-residue, gas-phase hydrogen deuterium exchange (HDX) kinetics for individual amino acid residues on selected ion conformer types of the model peptide KKDDDDDIIKIIK have been examined using ion mobility spectrometry (IMS) and HDX-tandem mass spectrometry (MS/MS) techniques. The [M + 4H](4+) ions exhibit two major conformer types with collision cross sections of 418 Å(2) and 446 Å(2); the [M + 3H](3+) ions also yield two different conformer types having collision cross sections of 340 Å(2) and 367 Å(2). Kinetics plots of HDX for individual amino acid residues reveal fast- and slow-exchanging hydrogens. The contributions of each amino acid residue to the overall conformer type rate constant have been estimated. For this peptide, N- and C-terminal K residues exhibit the greatest contributions for all ion conformer types. Interior D and I residues show decreased contributions. Several charge state trends are observed. On average, the D residues of the [M + 3H](3+) ions show faster HDX rate contributions compared with [M + 4H](4+) ions. In contrast the interior I8 and I9 residues show increased accessibility to exchange for the more elongated [M + 4H](4+) ion conformer type. The contribution of each residue to the overall uptake rate showed a good correlation with a residue hydrogen accessibility score model calculated using a distance from charge site and initial incorporation site for nominal structures obtained from molecular dynamic simulations (MDS).

2D IR cross peaks reveal hydrogen-deuterium exchange with single residue specificity.

A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

Apolipoprotein A-I helical structure and stability in discoidal high-density lipoprotein (HDL) particles by hydrogen exchange and mass spectrometry

To understand high-density lipoprotein (HDL) structure at the molecular level, the location and stability of α-helical segments in human apolipoprotein (apo) A-I in large (9.6nm) and small (7.8nm) discoidal HDL particles were determined by hydrogen-deuterium exchange (HX) and mass spectrometry methods. The measured HX kinetics of some 100 apoA-I peptides specify, at close to amino acid resolution, the structural condition of segments throughout the protein sequence and changes in structure and stability that occur on incorporation into lipoprotein particles. When incorporated into the large HDL particle, the nonhelical regions in lipid-free apoA-I (residues 45-53, 66-69, 116-146, and 179-236) change conformation from random coil to α-helix so that nearly the entire apoA-I molecule adopts helical structure (except for the terminal residues 1-6 and 237-243). The amphipathic α-helices have relatively low stability, in the range 3-5kcal/mol, indicating high flexibility and dynamic unfolding and refolding in seconds or less. A segment encompassed by residues 125-158 exhibits bimodal HX labeling indicating co-existing helical and disordered loop conformations that interchange on a time scale of minutes. When incorporated around the edge of the smaller HDL particle, the increase in packing density of the two apoA-I molecules forces about 20% more residues out of direct contact with the phospholipid molecules to form disordered loops, and these are the same segments that form loops in the lipid-free state. The region of disc-associated apoA-I that binds the lecithin-cholesterol acyltransferase enzyme is well structured and not a protruding unstructured loop as reported by others.

Hydrogen Exchange Mass Spectrometry of Bacteriorhodopsin Reveals Light-Induced Changes in the Structural Dynamics of a Biomolecular Machine

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.

Light-Controlled Protein Dynamics Observed with Neutron Spin Echo Measurements

A photoresponsive surfactant has been used as a means to control protein structure and dynamics with light illumination. This cationic azobenzene surfactant, azoTAB, which undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, adopts a relatively hydrophobic, trans structure under visible light illumination and a relatively hydrophilic cis structure under UV light illumination. Small-angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy were used to measure the tertiary structure and internal dynamics of lysozyme in the presence of the photosurfactant, respectively. The SANS-based in vitro structures indicate that under visible light the photosurfactant induces partial unfolding that principally occurs away from the active site near the hinge region connecting the α and β domains. Upon UV exposure, however, the protein refolds to a nativelike structure. At the same time, enhanced internal dynamics of lysozyme were detected with the surfactant in the trans form through NSE measurements of the Q-dependent effective diffusion coefficient (D(eff)) of the protein. In contrast, the D(eff) values of lysozyme in the presence of cis azoTAB largely agree with the rigid-body calculation as well as those measured for pure lysozyme, suggesting that the native protein is dormant on the nanosecond time and nanometer length scales. Lysozyme internal motions were modeled by assuming a protein of two (α and β domains) or three (α and β domains and the hinge region) domains connects by either soft linkers or rigid, freely rotating bonds. Protein dynamics were also tracked with Fourier transform infrared spectroscopy through hydrogen-deuterium exchange kinetics, which further demonstrated enhanced protein flexibility induced by the trans form of the surfactant relative to the native protein. Ensemble-averaged intramolecular fluorescent resonance energy transfer measurements similarly demonstrated the enhanced dynamics of lysozyme with the trans form of the photosurfactant. Previous results have shown a significant increase in protein activity in the presence of azoTAB in the trans conformation. Combined, these results provide insight into a unique light-based method of controlling protein structure, dynamics, and function and strongly support the relevance of large domain motions for the activity of proteins.