Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles.

TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein. We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased. Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax ∼95 Å in DPC alone versus ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (Kd ∼70 µM in DPC alone versus ∼0.91 µM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST). In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.

Analysis and modeling of SDS and DPC micelle SAXS data for membrane protein solution structure characterization.

Herein, we present analysis and analytical modeling of Small Angle X-ray Scattering (SAXS) data on two surfactants forming micelles (i.e., sodium dodecyl sulfate and dodecyl phosphocholine) and used for the study in solution of mTSPO, the translocator membrane protein from Mus musculus, as supporting data of the research article published in Biochimie (Combet et al., 2022). For both surfactants, concentration series were measured at two Synchrotron SAXS-beamlines. After reduction, buffer subtraction and water calibration of the data, SAXS curves were normalized to surfactant concentration to highlight possible changes in micelle shape or presence of inter-micellar weak interactions. They were then analyzed in terms of radius of gyration (R G), absolute forward intensity (I0) to access the surfactant aggregation number (Na ) and pair-distance distribution function (P(r)), which gives information on the shape and dimensions of the micelles. Finally, an analytical modeling using SasView - a SAS analysis software package (https://www.sasview.org/) - was performed to describe structural features of the two surfactant micelles at a concentration at which no change in the micelle shape nor weak interactions are observed. A core-shell ellipsoidal model was used to fit the SAXS curves, which provided geometrical parameters of the micelles (equatorial and polar radii, shell thickness) and also scattering length densities (SLD) of both the hydrophobic core and the hydrophilic shell. Hydration of polar heads into the micelle shell could be estimated from micelle volume calculations (V core and V shell). These parameters are particularly useful when modeling SAXS curves of membrane protein-surfactant complexes as described in Combet et al. (2022).

Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein.

The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation. Here, we decipher the solution structures of the recombinant mTSPO without ligand both in (i) SDS, the detergent used to extract and purify the protein from E. coli inclusion bodies, and (ii) DPC, the detergent used to solve the PK 11195-binding mTSPO NMR structure. We report partially refolded and less flexible mTSPO helices in DPC compared to SDS. Besides, DPC stabilizes the tertiary structure of mTSPO, as shown by a higher intrinsic Trp fluorescence and changes in indole environment. We evaluate by SEC-MALLS that ∼135 SDS and ∼100 DPC molecules are bound to mTSPO. SEC-small-angle X-ray (SAXS) and neutron (SANS) scattering confirm a larger mTSPO-detergent complex in SDS than in DPC. Using the contrast-matching technique in SEC-SANS, we demonstrate that mTSPO conformation is more compact and less flexible in DPC than in SDS. Combining ab initio modeling with SANS, we confirm that mTSPO conformation is less elongated in DPC than in SDS. However, the free-ligand mTSPO envelope in DPC is not as compact as the PK 11195-binding protein NMR structure, the ligand stiffening the protein.

Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations.

Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-ß-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM.

From Electronic Waste to Suzuki-Miyaura Cross-Coupling Reaction in Water: Direct Valuation of Recycled Palladium in Catalysis.

Invited for this month's cover is the group of Damien Bourgeois at the Marcoule Institute for Separation Chemistry (ICSM). The image shows how a short process chain can efficiently transform our waste into an active catalyst. The Full Paper itself is available at 10.1002/cssc.202001155.

From Electronic Waste to Suzuki-Miyaura Cross-Coupling Reaction in Water: Direct Valuation of Recycled Palladium in Catalysis.

From electronic waste to Pd-catalyzed reaction! The straightforward valuation of palladium recovered from electronic waste is reported here. Following a classical leaching stage, palladium is selectively extracted from a complex aqueous mixture of metallic cations into an organic phase. Afterwards, the judicious choice of a surfactant enables stabilization of palladium during back extraction cycles, and the direct preparation of an aqueous micellar solution, which can be employed in a model Suzuki-Miyaura cross-coupling reaction. Clean phase separation is observed, and distribution of all components between organic and aqueous phases is mastered. The proposed process avoids several waste generating steps dedicated to palladium isolation and ultimate purification, as well as the preparation of palladium pre-catalyst. This novel approach enables a better use of both natural resources and industrial wastes, through new cycles in circular economy.

Biotinylated non-ionic amphipols for GPCR ligands screening.

We present herein the synthesis of biotin-functionalized polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG)8-alkyne that resulted in formation of the biotinylated polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized polymers was done using both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based on their ability to bind the immobilized receptor. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity.

Despite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57)2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an "extremely open" N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.

Fluorinated diglucose detergents for membrane-protein extraction.

Fluorinated surfactants have scarcely been explored for the direct extraction of proteins from membranes because fluorination is believed to abrogate detergency. However, we have recently shown that a commercially available fluorinated surfactant readily solubilizes lipid membranes, thereby suggesting that fluorination per se does not interfere with detergent activity. In this work, we developed new fluorinated surfactants that exhibit detergency in terms of both lipid-vesicle solubilization and membrane-protein extraction. The compounds made and tested contain two glucose moieties as polar headgroup, a hydrogenated thioether linker, and a perfluorinated alkyl tail with either 4, 6, or 8 carbon atoms. The physicochemical properties of the micelles formed by the three fluorinated surfactants were evaluated by NMR spectroscopy, surface tensiometry, isothermal titration calorimetry, dynamic light scattering, small-angle X-ray scattering, and analytical ultracentrifugation. At 25 °C, micellization was mainly entropy-driven, and the CMC values were found to decrease with chain length of the fluorinated tail, whereas the aggregation number increased with chain length. Remarkably, all three surfactants were found to solubilize lipid vesicles and extract a broad range of proteins from Escherichia coli membranes. These findings demonstrate, for the first time, that nonionic fluorinated surfactants could be further exploited for the direct extraction and solubilization of membrane proteins.

The stress sigma factor of RNA polymerase RpoS/σS is a solvent-exposed open molecule in solution.

In bacteria, one primary and multiple alternative sigma (σ) factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (Eσ) with different promoter recognition specificities. The alternative σ factor RpoS/σS is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free σ factor remains elusive. The current model suggests that extensive interdomain contacts in a free σ factor result in a compact conformation that masks the DNA-binding determinants of σ, explaining why a free σ factor does not bind double-stranded promoter DNA efficiently. Here, we explored the solution conformation of σS using amide hydrogen/deuterium exchange coupled with mass spectrometry, NMR, analytical ultracentrifugation and molecular dynamics. Our data strongly argue against a compact conformation of free σS Instead, we show that σS adopts an open conformation in solution in which the folded σ2 and σ4 domains are interspersed by domains with a high degree of disorder. These findings suggest that E binding induces major changes in both the folding and domain arrangement of σS and provide insights into the possible mechanisms of regulation of σS activity by its chaperone Crl.

Characterization of New Detergents and Detergent Mimetics by Scattering Techniques for Membrane Protein Crystallization.

Membrane proteins are difficult to manipulate and stabilize once they have been removed from their native membranes. However, despite these difficulties, successes in membrane-protein structure determination have continued to accumulate for over two decades, thanks to advances in chemistry and technology. Many of these advances have resulted from efforts focused on protein engineering, high-throughput expression, and development of detergent screens, all with the aim of enhancing protein stability for biochemistry and biophysical studies. In contrast, considerably less work has been done to decipher the basic mechanisms that underlie the structure of protein-detergent complexes and to describe the influence of detergent structure on stabilization and crystallization. These questions can be addressed using scattering techniques (employing light, X-rays, and/or neutrons), which are suitable to describe the structure and conformation of macromolecules in solution, as well as to assess weak interactions between particles, both of which are clearly germane to crystallization. These techniques can be used either in batch modes or coupled to size-exclusion chromatography, and offer the potential to describe the conformation of a detergent-solubilized membrane protein and to quantify and model detergent bound to the protein in order to optimize crystal packing. We will describe relevant techniques and present examples of scattering experiments, which allow one to explore interactions between micelles and between membrane protein complexes, and relate these interactions to membrane protein crystallization.

Innovative High-Throughput SAXS Methodologies Based on Photonic Lab-on-a-Chip Sensors: Application to Macromolecular Studies.

The relevance of coupling droplet-based Photonic Lab-on-a-Chip (PhLoC) platforms and Small-Angle X-Ray Scattering (SAXS) technique is here highlighted for the performance of high throughput investigations, related to the study of protein macromolecular interactions. With this configuration, minute amounts of sample are required to obtain reliable statistical data. The PhLoC platforms presented in this work are designed to allow and control an effective mixing of precise amounts of proteins, crystallization reagents and buffer in nanoliter volumes, and the subsequent generation of nanodroplets by means of a two-phase flow. Spectrophotometric sensing permits a fine control on droplet generation frequency and stability as well as on concentration conditions, and finally the droplet flow is synchronized to perform synchrotron radiation SAXS measurements in individual droplets (each one acting as an isolated microreactor) to probe protein interactions. With this configuration, droplet physic-chemical conditions can be reproducibly and finely tuned, and monitored without cross-contamination, allowing for the screening of a substantial number of saturation conditions with a small amount of biological material. The setup was tested and validated using lysozyme as a model of study. By means of SAXS experiments, the proteins gyration radius and structure envelope were calculated as a function of protein concentration. The obtained values were found to be in good agreement with previously reported data, but with a dramatic reduction of sample volume requirements compared to studies reported in the literature.

Coupling High Throughput Microfluidics and Small-Angle X-ray Scattering to Study Protein Crystallization from Solution.

In this work, we propose the combination of small-angle X-ray scattering (SAXS) and high throughput, droplet based microfluidics as a powerful tool to investigate macromolecular interactions, directly related to protein solubility. For this purpose, a robust and low cost microfluidic platform was fabricated for achieving the mixing of proteins, crystallization reagents, and buffer in nanoliter volumes and the subsequent generation of nanodroplets by means of a two phase flow. The protein samples are compartmentalized inside droplets, each one acting as an isolated microreactor. Hence their physicochemical conditions (concentration, pH, etc.) can be finely tuned without cross-contamination, allowing the screening of a huge number of saturation conditions with a small amount of biological material. The droplet flow is synchronized with synchrotron radiation SAXS measurements to probe protein interactions while minimizing radiation damage. To this end, the experimental setup was tested with rasburicase (known to be very sensitive to denaturation), proving the structural stability of the protein in the droplets and the absence of radiation damage. Subsequently weak interaction variations as a function of protein saturation was studied for the model protein lysozime. The second virial coefficients (A2) were determined from the X-ray structure factors extrapolated to the origin. A2 obtained values were found to be in good agreement with data previously reported in literature but using only a few milligrams of protein. The experimental results presented here highlight the interest and convenience of using this methodology as a promising and potential candidate for studying protein interactions for the construction of phase diagrams.

Use of dynamic light scattering and small-angle X-ray scattering to characterize new surfactants in solution conditions for membrane-protein crystallization.

The structural and interactive properties of two novel hemifluorinated surfactants, F2H9-ß-M and F4H5-ß-M, the syntheses of which were based on the structure and hydrophobicity of the well known dodecyl-ß-maltoside (DD-ß-M), are described. The shape of their micellar assemblies was characterized by small-angle X-ray scattering and their intermicellar interactions in crystallizing conditions were measured by dynamic light scattering. Such information is essential for surfactant phase-diagram determination and membrane-protein crystallization.

Micellar and biochemical properties of a propyl-ended fluorinated surfactant designed for membrane-protein study.

Our goal is to design optimised fluorinated surfactants for handling membrane proteins in solution. We report herein the self-assembling and biochemical properties of a new hemifluorinated surfactant (H3F6H3DigluM) with a branched diglucosylated polar head group and an apolar tail consisting of a perfluorohexane core decorated with a hydrogenated propyl tip. For the sake of comparison, its fluorinated analogue without propyl tip (F6H3DigluM) was also studied. Isothermal titration calorimetry and surface tension showed that the addition of a propyl tip has a significant effect on the overall hydrophobicity of the surfactant, in contrast to the behaviour described when adding an ethyl tip to a fluorinated surfactant. From dynamic light scattering, analytical ultracentrifugation and small-angle X-ray scattering, both H3F6H3DigluM and F6H3DigluM self-assemble into small globular micelles of 5-7 nm in diameter and have aggregation numbers of 62±8 and 46±2, respectively. Finally, H3F6H3DigluM was found to be the best fluorinated surfactant developed in our group to stabilise the model membrane protein bacteriorhodopsin (bR) in aqueous solution. This study demonstrates the suitability of this new propyl-ended fluorinated surfactant for biochemical and structural applications and confirms the superiority of hemifluorinated chains over fluorinated ones.

Influence of hydrophobic micelle structure on crystallization of the photosynthetic RC-LH1-PufX complex from Rhodobacter blasticus.

Small angle X-ray scattering (SAXS) experiments are performed on two non-ionic surfactants, the dodecyl ß-maltoside (DDßM) and the propyl(bi)cyclohexyl α-maltoside (PCCαM), a maltoside derivative containing a rigid bicyclohexyl group as hydrophobic chain, in order to compare the influence of both hydrophobic moiety structure and anomeric form on micelle form factors and intermicellar interactions relevant for membrane protein crystallization. Density and refractive index measurements were performed in order to determine volumetric and optical properties of surfactants, essential for determination of micelle molar masses by both SAXS and SEC-MALLS. SAXS form factors were analyzed by Guinier approximation and inverse Fourier transformation, to obtain the radius of gyration (RG) and the pair distribution function (P(r)) of each surfactant. Form factor model fitting was also performed to describe the shape and the assembly of both surfactant micelles. Finally, second virial coefficients were measured at different percentages of polyethylene glycol 3350, in order to correlate surfactant intermicellar interactions and RC-LH1-PufX phase diagram. It is thus found that while size, shape, and dimensions of micelles are slightly similar for both surfactants, their molar mass and aggregation number differ significantly. PCCαM are more densely packed than DDßM, which reflects (1) an increase in van der Waals contacts between PCCαM hydrophobic chains in the micelle bulk and (2) a supplementary intermicellar attraction compared to DDßM. Finally addition of PEG, which induces a depletion attraction, decreases the solubility of the RC-LH1-PufX complex in PCCαM.

A new high-performance thin layer chromatography-based assay of detergents and surfactants commonly used in membrane protein studies.

The hydrophobic nature of membrane proteins (MPs) necessitates the use of detergents for their extraction, solubilization and purification. Because the concentration of amphiphiles is crucial in the crystallization process, detergent quantification is essential to routine analysis. Here we describe a quantitative high-performance thin-layer chromatography (HPTLC) method we developed for the detection of small quantities of detergent bound to solubilized MPs. After optimization of aqueous deposit conditions, we show that most detergents widely used in membrane protein crystallography display distinctive mobilities in a mixture of dichloromethane, methanol and acetic acid 32:7.6:0.4 (v/v/v). Migration and derivatization conditions were optimized with n-dodecyl-ß-D-maltoside (DDM), the most popular detergent for membrane protein crystallization. A linear calibration curve very well fits our data from 0.1 to 1.6 µg of DDM in water with a limit of detection of 0.05 µg. This limit of detection is the best achieved to date for a routine detergent assay, being not modified by the addition of NaCl, commonly used in protein buffers. With these chromatographic conditions, no prior treatment is required to assess the quantities of detergent bound to purified MPs, thus enabling the quantification of close structure detergents via a single procedure. This HPTLC method, which is fast and requires low sample volume, is fully suitable for routine measurements.

Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

BACKGROUND: Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. CONCLUSIONS: The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process.

A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization.

Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-ß-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b(6 )f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.

Structure-function perturbation and dissociation of tetrameric urate oxidase by high hydrostatic pressure.

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.