High-pressure Macromolecular Crystallography Research Articles

The Promise and Challenge of High Pressure Macromolecular Crystallography

Since its introduction in the early 1970s, high pressure crystallography (HPX) has shown great potential for the investigation of different types of matter. Using diamond anvil cells, HPX is an emerging technique that has been rapidly implemented, making it available to biologists, and there is immense potential for utilizing this technique in biological systems in the future. At the molecular level, high-pressure crystallographic investigation provides information on structural characteristics that not only determine the native conformation of a protein but also the conformations with higher free-energy, thus revealing function-related structural changes and properties that can be modified as a result of pressurization. The increase in the number of crystal structures of different macromolecules determined under high pressure over the last five decades can be ascribed mainly to two factors: the emergence of high-pressure cells with very large, open angles, and the advent of third generation synchrotron sources. The use of high pressure crystallography as a research tool has been shown to contribute to the advancements in the basic fields of biochemistry (protein misfolding and aggregation), biophysics (protein stability), and biotechnology (food processing). Presently, with a growing interest in biomedicine and nanotechnology, this nonstandard method appears to be a valid instrument for probing more challenging and complex systems. In this review, we present the method, highlight a selection of recent applications, and describe challenges for high pressure macromolecular crystallography (HPMX).

Exploring the structural dynamics of proteins by pressure perturbation using macromolecular crystallography.

High pressure is a convenient thermodynamic parameter to probe the dynamics of proteins as it is intimately related to volume which is essential for protein function. To be biologically active, a protein fluctuates between different substates. Pressure perturbation can promote some hidden substates by modifying the population between them. High pressure macromolecular crystallography (HPMX) is a perfect tool to capture and to characterize such substates at a molecular level providing new insights on protein dynamics. The present chapter describes the use of the diamond anvil cell to perform HPMX experiments. It also provides tips on sample preparation and optimal data collection as well as on efficient analysis of the resulting high-pressure structures.

Equilibria between conformational states of the Ras oncogene protein revealed by high pressure crystallography.

In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg2+·GppNHp and Ras(D33K)·Mg2+·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such in crystallo phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.

A high-pressure macromolecular crystallography capability developed at CHESS

A high-pressure macromolecular crystallography capability developed at CHESS

Functional Sub-states by High-pressure Macromolecular Crystallography.

At the molecular level, high-pressure perturbation is of particular interest for biological studies as it allows trapping conformational substates. Moreover, within the context of high-pressure adaptation of deep-sea organisms, it allows to decipher the molecular determinants of piezophily. To provide an accurate description of structural changes produced by pressure in a macromolecular system, developments have been made to adapt macromolecular crystallography to high-pressure studies. The present chapter is an overview of results obtained so far using high-pressure macromolecular techniques, from nucleic acids to virus capsid through monomeric as well as multimeric proteins.

High Pressure Macromolecular Crystallography.

X-ray crystallography is a powerful tool for the high resolution structural study of biomacromolecules. One of the most important beneficial feature of the method is the possibility of the direct observation of hydration water structure. In recent years, significant development in high-pressure macromolecular crystallography (HPMX) using a diamond anvil cell (DAC) has been performed in combination with shorter wavelength X-ray of synchrotron radiation. The number of protein structures determined by HPMX at pressure ranging from several hundred MPa to 1 GPa is gradually growing. In this chapter, we describe DAC with its usage, and then an example of HPMX study on hydration structure of 3-isopropylmalate dehydrogenase (IPMDH).

Structural study of the pressure adaptation of proteins from deep-sea bacteria

In recent years, significant development in the high-pressure macromolecular crystallography (HPMX) using a diamond anvil cell (DAC) has been performed especially by Prof. R. Fourme's group in combination with shorter wavelength X-ray of synchrotron radiation [1]. We are also trying to establish HPMX experimental environment at the Photon Factory, Japan [2]. HPMX is a unique method that provides high-resolution structural informations under pressure including hydration waters at a molecular surface and an internal cavity. One of the important applications is studying functional sub-states of biological macromolecules, and we are attempting to elucidate a mechanism of pressure tolerance of proteins from several organisms living in deep seas such as the Mariana Trench. For example, 3-isopropylmalate dehydrogenase (IPMDH) from the deep-sea bacterium Shewanella benthica DB21MT-2 is much more tolerant to the pressure stress than its counterpart from the land bacterium S. oneidensis MR-1 (So-IPMDH), even though these two enzymes share about 85% amino-acid identity. Crystal structures of So-IPMDH have been determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa. Waters penetrating into the internal cavity at the dimer interface and squeezing into a molecular surface cleft opposite the active site are observed at above 410 MPa and 580 MPa, respectively [3]. The bottom of the cleft of So-IPMDH is characterized by the presence of Ser266 at the bottom, which is able to form a hydrogen bond to the squeezed water molecule. On the other hand, IPMDHs from deep-sea bacterium favors an alanine at the same position (Ala266). As expected, no water penetration is observed there at the same pressure range for the S266A mutated So-IPMDH, and the mutation develops tolerance to the pressure. In addition, some results of the high-pressure structure analysis of other proteins, and pressure-induced phase transitions in some protein crystals will also be mentioned.

Pressure and high-pressure macromolecular crystallography at the ESRF

Almost all diffraction experiments in structural biology are done at cryogenic temperature to mitigate radiation damage with cryoprotected crystals to avoid solvent crystallization and its harmful consequences: formation of ice rings, loss of diffraction and exaggeration of mosaicities. Crystallographers often protect crystals with glycerol rather than with genuine conditions whose search is time consuming. Moreover, cryo-protection has detrimental consequences: Soaking somewhat destabilizes crystals, mosaicity expend upon cooling, cryo-agents may interact with macromolecules and in extreme cases, cryo-conditions are destructive. High pressure (HP) cooling is an alternative method which consists of flash-cooling cryoprotectant-free samples under 200 MPa of Helium [1]. The solvent is directly turned into high density amorphous (HDA) ice avoiding water crystallization and preserving/improving the sample quality owing to the absence of cryo-agents and to HAD-ice properties. We have developed a HP-cooling system and its associated methodology paying attention to user-friendliness and throughput aspects through a new pluggable Spine compatible base-pin and to its handling-toolkit [2]. The machine allows vitrifying solution with 5% glycerol for cooling diluted bio-objects, but importantly, it is very effective for cryoprotectant-free crystals since the sole mother liquor components act as anti-freeze agents. Its capability was demonstrated with test crystals and resulting structures appear isomorphous to those deposited in the PDB since the structural changes are limited to flexible loops. Nevertheless, pressure is a key thermodynamic variable which produces structural modifications associated with different conformational protein substates or reaction coordinates [3], and we provide examples of HP structures which address biological questions. On the basis of the HP-cooling technique, we have designed a novel of pressure cell for gases of biological interest. The method takes advantage of thermodynamic properties which allow liquefying pressurized gases; crystals are soaked in the gas phase prior to be flash-frozen in the liquid phase without pressure relaxation. This technique has been applied to noble gas derivatives and oxygen sensitive macromolecules.

High-pressure macromolecular crystallography and NMR: status, achievements and prospects

Biomacromolecules are thermodynamic entities that exist in general as an equilibrium mixture of the basic folded state and various higher-energy substates including all functionally relevant ones. Under physiological conditions, however, the higher-energy substates are usually undetectable on spectroscopy, as their equilibrium populations are extremely low. Hydrostatic pressure gives a general solution to this problem. As proteins generally have smaller partial molar volumes in higher-energy states than in the basic folded state, pressure can shift the equilibrium toward the former substantially, and allows their direct detection and analysis with X-ray crystallography or NMR spectroscopy at elevated pressures. These techniques are now mature, and their status and selected applications are presented with future prospects.

High pressure macromolecular crystallography for structural biology: a review

Abstract In recent years, significant progress in high pressure macromolecular crystallography has been observed. It can be attributed both to the developments in experimental techniques, as well as to recognition of importance of high pressure protein studies in biochemistry and biophysics. The number of protein structures determined at pressure up to 1 GPa is growing. The unique advantages of this method can greatly improve the investigation of higher energy conformers of functional significance and our understanding of functionally important conformers, protein folding processes and the structural base of conformational diseases.

Macromolecular crystallography at high pressure with pneumatic diamond anvil cells handled by a six-axis robotic arm

A new pneumatic diamond anvil cell has been constructed, generating continuous pressure and temperature variations in the range 0–2.5 GPa and 293–393 K. The cell is designed mainly for high-pressure macromolecular crystallography and should facilitate pressure and temperature annealing of the sample. The analysis is reported of several diffraction data sets of tetragonal hen egg-white lysozyme crystals loaded either in the new cell or in a currently used membrane-based cell. These experiments were performed on beamline FIP-BM30A at the ESRF, Grenoble, a macromolecular crystallography beamline on a bending magnet. Cells were handled and automatically centred by a six-axis robotic arm that was used as a goniometer for data collection by the oscillation method.

Isothermal compressibility of macromolecular crystals and macromolecules derived from high-pressure X-ray crystallography

The compressibility of several nucleic acid and globular protein crystals has been investigated by high-pressure macromolecular crystallography. Further, crystal structures at four different pressures allowed the determination of the intrinsic compressibilityversuspressure of d(GGTATACC)2and hen egg-white lysozyme. For lysozyme, the values for the intrinsic molecular compressibility at atmospheric pressure and the nonlinearity index were 0.070 GPa−1and 8.15, respectively. On the basis of two crystal structures at atmospheric and high pressure, similar, albeit less complete, information was derived for d(CGCGAATTCGCG)2and bovine erythrocyte Cu,Zn superoxide dismutase. Using these data and accurate calculations of the solvent-excluded volume, the apparent solvent compressibility in the crystalline state was determined as a function of pressure and compared with results from a simple model that assumes invariant unit-cell content, with the conclusion that solvent compressibility was abnormal for three out of the five crystals investigated. Experimental results suggest that macromolecular crystals submitted to high pressure may have a variable unit-cell mass due to solvent exchange with the surrounding pool, as already observed in other hydrated crystals such as zeolites.

Advances in High-Pressure Biophysics: Status and Prospects of Macromolecular Crystallography

A survey of the main interests of high pressure for molecular biophysics highlights the possibility of exploring the whole conformational space using pressure perturbation. A better understanding of fundamental mechanisms responsible for the effects of high pressure on biomolecules requires high-resolution molecular information. Thanks to recent instrumental and methodological progress taking advantage of the remarkable adaptation of the crystalline state to hydrostatic compression, pressure-perturbed macromolecular crystallography is now a full-fledged technique applicable to a variety of systems, including large assemblies. This versatility is illustrated by selected applications, including DNA fragments, a tetrameric protein, and a viral capsid. Binding of compressed noble gases to proteins is commonly used to solve the phase problem, but standard macromolecular crystallography would also benefit from the transfer of experimental procedures developed for high-pressure studies. Dedicated short-wavelength synchrotron radiation beamlines are unarguably required to fully exploit the various facets of high-pressure macromolecular crystallography.

Salting-In Effects on Urate Oxidase Crystal Design

In this paper, solubility and interactions in solution of the recombinant urate oxidase from Aspergillus flavus, rasburicase, are studied both in the absence and in the presence of salt at a pH close to the pI. An intense salting-in effect is demonstrated first by an increased solubility when various salts are added. Thus, merely adding salt does not induce rasburicase crystallization. Second virial coefficient measurements also confirm this effect by exhibiting repulsive interactions over a large range of salt concentrations. Therefore, the salting-in effect enables the stabilization of rasburicase solution at high concentrations. Moreover, it enables crystals of improved size and habit to be grown when polymer is added to a solution of rasburicase concentrated with salt, or when salt is removed from it. We also show, with the example of high pressure macromolecular crystallography, that salt enables the stabilization of the desired polymorph under the highly concentrated polyethylene glycol conditions required by this technique.

High-pressure macromolecular crystallography: status, applications and prospects

High-pressure macromolecular crystallography: status, applications and prospects

Toward fully fledged high-pressure macromolecular crystallography

Pressure allows one to explore the energy landscape and the various conformations of macromolecules. This might be one of the most interesting prospects of combining macromolecular crystallography (MX) with pressure perturbation. This paper presents innovations in high-pressure macromolecular crystallography (HPMX). A new-generation diamond anvil cell extends the technical feasibility of HPMX experiments up to about 2.5 GPa (depending on the sample). Thanks to the large useful aperture (82°) provided by this cell and special loading techniques (use of splinter and/or multiple samples), HPMX can now be more readily applied to crystals with very anisotropic habits and/or low-symmetry space groups, as demonstrated by analysis of diffraction data collected at 140 MPa from orthorhombic urate oxidase crystals. The behaviour of hexagonal crystals of an octanucleotide was investigated up to 2 GPa.

High pressure macromolecular crystallography: The 140-MPa crystal structure at 2.3 Å resolution of urate oxidase, a 135-kDa tetrameric assembly

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 Å resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.

A new dimension in structural biology: fully fledged high-pressure macromolecular crystallography

The amount of accurate crystallographic data currently available on macromolecular structures at high pressure is extremely limited, mainly due to the lack of appropriate instrumentation and X-ray sources. A technical breakthrough has been achieved with a set-up at the ESRF ID30 beamline equipped with a diamond anvil cell and a large imaging plate, and taking advantage from undulators providing a quasi-plane wave of ultrashort wavelength X-rays. The accessible pressure range is increased by nearly one order of magnitude with respect to beryllium cells. A nearly perfect long-range order is preserved in protein and virus crystals compressed below the denaturation pressure. The quality of diffraction data collected at high pressure can meet usual standards, especially in the case of high symmetry space groups. An overview of main results and ongoing studies is given, including the 7 kbar structure of hen egg-white lysozyme refined at 1.6 Å resolution and structural investigations of cowpea mosaic virus (CPMV), the first macromolecular assembly investigated at high pressure by single crystal X-ray diffraction. The ordering effect of high pressure on the molecular packing of disordered P23 crystals was highlighted. The 2.8 Å resolution structure of CPMV at 3.3 kbar was fully refined using high completeness data collected on pressure-ordered I23 crystals.