Cryogenic X-ray Crystallography Research Articles

Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography.

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.

In crystallo thermodynamic analysis of conformational change of the topaquinone cofactor in bacterial copper amine oxidase

In the catalytic reaction of copper amine oxidase, the protein-derived redox cofactor topaquinone (TPQ) is reduced by an amine substrate to an aminoresorcinol form (TPQamr), which is in equilibrium with a semiquinone radical (TPQsq). The transition from TPQamr to TPQsq is an endothermic process, accompanied by a significant conformational change of the cofactor. We employed the humid air and glue-coating (HAG) method to capture the equilibrium mixture of TPQamr and TPQsq in noncryocooled crystals of the enzyme from Arthrobacter globiformis and found that the equilibrium shifts more toward TPQsq in crystals than in solution. Thermodynamic analyses of the temperature-dependent equilibrium also revealed that the transition to TPQsq is entropy-driven both in crystals and in solution, giving the thermodynamic parameters that led to experimental determination of the crystal packing effect. Furthermore, we demonstrate that the binding of product aldehyde to the hydrophobic pocket in the active site produces various equilibrium states among two forms of the product Schiff-base, TPQamr, and TPQsq, in a pH-dependent manner. The temperature-controlled HAG method provides a technique for thermodynamic analysis of conformational changes occurring in protein crystals that are hardly scrutinized by conventional cryogenic X-ray crystallography.

New Structural Analysis Methods for Hemoglobin Crystals. Time-Resolved Cryogenic X-Ray Crystallography with Extended Pulsed-Laser Pumping and 3D Imaging by X-Ray Fluorescence Holography

Hemoglobin (Hb) has long served as a model for studying protein allostery in general. Static crystal structures of deoxy tense (T) state and oxy relaxed (R) state of Hb reveal multiple differences at both tertiary and quaternary levels. However, we still lack an adequate understanding of how these structural differences control ligand binding. Here we present two methods for Hb crystals to investigate the ligation-linked protein motion and the atomic details of the heme active site structure.The first method is time-resolved X-ray crystallography combined with high-repetition pulsed-laser pumping and cryogenic trapping. At cryogenic temperatures under 50 K, a photo-dissociated ligand molecule can move to only the limited range near the heme, and the protein moiety can undergo only small structural changes. By contrast, at 100-140 K under continuous pulsed-laser irradiation, we found that ligand migration and protein relaxation can be observed. We have succeeded in tracking the photo-dissociated ligand molecule that migrates along the pathway connecting inner cavities of Hb in a different direction in each subunit.The second method is atomic-resolution X-ray fluorescence holography (XFH), which utilizes fluorescing atoms as a wave source within a crystal sample. So far, XFH has generally been applied to inorganic materials to investigate the microscopic environments of trace metals. This is the first application of XFH for protein crystals. For Hb crystals, XFH can provide the three dimensional atomic images around the iron central atoms of the individual hemes. We have carried out XFH measurements at 100 K of CO-bound Hb crystals at BL6C, KEK, Japan. We will present our latest data and share with you the results and discussion of this work.

Probability Distributions of Hydration Water Molecules around Polar Protein Atoms Obtained by a Database Analysis

Hydration structures on protein surfaces are visualized by high-resolution cryogenic X-ray crystallography. We calculated the probability distributions of 4,831,570 hydration water molecules found around the 4,214,227 polar atoms in main chains and hydrophilic side chains from the 17,984 crystal structures in the Protein Data Bank. The structures are refined using the diffraction data collected below 150 K and at resolutions of better than 2.2 A. The calculated distributions were nonrandom but condensed into a few clusters. The clusters were decomposed into the distance and angular distributions by viewing from the polar coordinate system. The major peaks in the clusters were almost located along the directions of the N-H and O-H bonds or the lone pairs of oxygen atoms. The Gaussian fitting method was applied for the distribution profiles to evaluate quantitatively the peak positions and the widths. The parameters characterizing the distributions apparently depended on the hydrogen-bond partners of water molecules and on the modes whether the water molecules acted as donors or acceptors of protons. This led to propose the different roles of NH(n) (n = 1, 3), OH, and CO groups in protein hydration and possible in protein-ligand and protein-protein interaction: While C horizontal lineO groups appear to control the H-bond distances, NH(n) groups likely limit the angular range of H-bonds. The OH groups have both characteristics. In addition, it was also demonstrated that polar protein atoms were arranged to satisfy the tetrahedral hydrogen-bond geometry of water molecules, suggesting essential roles of water molecules in the folding process and in the stabilization of protein structures. These probability distributions are probably one of fundamental data to better understand the roles of hydration water molecules in the folding process and the stability of proteins in solution.

Investigation of Hydration Structures of Proteins

Water is a mother liquid of life. To understand why water is indispensable to life, I have been investigating the structures and interactions at protein-water interface, i.e. the hydration structures of proteins, by cryogenic X-ray crystallography and molecular dynamics simulation. Through developing devices and experimental techniques in cryogenic X-ray diffraction experiments, I realized that cryogenic X-ray crystallography is one of techniques effectively observe hydration structures of proteins surface. In addition, the author developed a novel calculation codes to characterize systematically hydration structures of the protein structures determined at around 100 K, as to the amount of water molecules, the hydrogen-bond geometries, the local and the global distribution of them on the surface of proteins. The standard tetrahedral hydrogen bonds of water molecules in bulk water were retained in the hydration structures and contributed to extend three-dimensional network of hydrogen bonds among hydration water molecules and oxygen and nitrogen atoms of protein surface. In cryo-crystallography of multi-subunit or multi-domain proteins, the reorganization of hydration structure occurred with the dynamical motions of proteins. In that, water molecules act as inhibitors for protein motions, glue to stabilize the higher-order structures or lubricant to realize conformational changes by utilizing its tetrahedral arms of hydrogen bonds. Thus, hydration water molecules must have great influences on the dynamics and functions of proteins in aqueous solution.

Structure of the Protein BPTI Derived with NOESY in Supercooled Water: Validation and Refinement of Solution Structures

Fidelity without frostbite: Refinement of an NMR solution structure of 6-kDa protein BPTI determined at 36 °C (see picture, red) with NOE distance constraints measured in supercooled water at −15 °C increased precision of backbone and core side-chain coordinates about twofold (blue). In contrast to cryogenic X-ray crystallography (−150 °C), supercooling to about −15 °C hardly affects the conformation of flexibly disordered surface side chains.

Water-mediated interaction at a protein–protein interface

Abstract The water-mediated indirect interaction between barnase and barstar was investigated by surface plasmon resonance measurement and cryogenic X-ray crystallography. Mutations of four acidic residues of barstar, D35A, D39A, E76A and E80A, decreased the binding free energies by 17.2, 25.2, 3.8 and 2.1 kJ mol−1, respectively, in the presence of 150 mM NaCl at pH 7.4 and 25 °C. The changes of the hydrated structures of the complexes caused by the mutations were localized around the mutational site, suggesting that difference in the binding free energy is closely correlated with difference in the local hydrated structure. Then, the averaged binding free energy was estimated at 4.4 kJ mol−1 per water-mediated indirect interaction on the basis of the difference in hydrogen bonding network.

An open-flow cryostat using helium gas for cryogenic x-ray diffraction experiments

An open-flow cryostat using helium gas has been developed for cryogenic x-ray crystallography. The device is composed of a cryostat and a heat-exchange apparatus, and dry nitrogen gas is supplied for shielding the cooled helium gas from atmosphere. The lowest temperature reached by the device is 35 K within a fluctuation of ±0.5 K at a flow rate of 6 l/min. This device is easy to operate and enables to study crystal structures and the temperature-dependent phenomena occurring in biological macromolecules and materials above 35 K.

Roles of hydration water molecules in molecular packing of the killer toxin from Pichia farinosa in its crystalline state investigated by cryogenic X-ray crystallography

The hydration structures around the killer toxin from Pichia farinosa were investigated by cryogenic X-ray crystallography. In particular, those contributing to the molecular association and the crystal contacts were analyzed with respect to the geometry and the networks of hydrogen bonds. The hydration water molecules attached on the surface so as to make up the surface shape in the contact complementary and mediated the intermolecular interactions through the networks of hydrogen bonds. Careful inspection of the contact area led to a proposal as to the molecular association mode of the toxin to determine the biological function in cells. In addition, the water-associated protein–protein interactions were approximated well by a simple theoretical equation on the solvation force expected in confined geometry. The present analysis may provide a way to analyze the crystal contact and molecular recognition in macromolecules in aqueous solution.

Structural Characteristics in Protein Hydration Investigated by Cryogenic X-ray Crystal Structure Analyses.

Cryogenic X-ray crystallography has heen applied to investigate thehydration structures of proteins. The amount of hydration water moleculesidentified at cryogenic temperature is more than twice those at ambienttemperature, and the structural models of proteins with a lot of hydrationwater molecules have provided much information to elucidate the static anddynamical characteristics of hydration structures of proteins. On proteinsurface, hydration water molecules distribute non-randomly and stillretain the tetrahedral hydrogen-bond geometry as well as in bulk solvent.In addition, water molecules form clathrate-like arrangements to cover thehydrophobic residues exposed to solvent. The standard interaction geometryenables the three-dimensional extension of hydrogen-bond networks aroundprotein molecules and, simultaneously, ensures the concerted reorganizationof hydration structures during the dynamical motion of proteins at work.The hydration structure analyses at cryogenic temperatures may contributeto understanding physical principles governing the dynamics of `molecularmachines' in aqueous environment.

水素結合における構造物性と機能 蛋白質の水和構造

Hydration structures surrounding protein molecules have great influences on folding, stability, physical properties and functions of the molecules. In spite of this fact, X-ray protein crystallography have not sufficiently contributed on hydration structure analyses of proteins. Recent progress in cryogenic techniques enables us to analyze hydration structures of proteins. Cryogenic analyses have provided the structural information on hydration patterns around hydrophobic residues and network structures formed by hydration water molecules. New insights into the hydration structures of proteins have been given by cryogenic X-ray crystallography.

Statistical Survey of Transition States and Conformational Substates of the Sperm Whale Myoglobin−CO Reaction System

The distribution of activation energies for ligand rebinding in conformational substates of sperm whale myoglobin is investigated with a molecular dynamics−quenching method. The technique approximately locates transition states for curve crossing processes using an interpolating Hamiltonian as in Marcus's electron transfer theory. Linear free energy relations are examined. The transition from the dissociated state to the activated state involves a collective motion of the protein. The activation energy is correlated more to the extent of the collective motion of the protein than to the change of any single bond or bond angle. The predicted average structures of fast and slow rebinding substates which will soon be accessible to determination by cryogenic X-ray crystallography are compared. The CO geometry of the activated protein complex from the simulation agrees well with the structure of a low temperature photodissociated intermediate determined by Schlichting et al.