In the last decade, significant breakthroughs have been occurred in the field of protein crystallization. These include advances in crystallization method, method of screening for crystallization conditions, precipitant agents and additves. Techniques developed for biochemical experiments have also contributed to protein crystallization. This paper gives an overview of methodological advances of protein crystallization method as well as future prospects in the method.
Synchrotron radiation has already become a common technique in the field of protein crystallogaphy. This review describes the properties of synchrotron radiation and instrumentations at the Photon Factory.
Selenomethionyl proteins are now widely used in protein crystallography. It can be used as one of the heavy atom derivatives having sufficient phasing power. In addition, the positions of selenium atoms can be easily determined through the use of difference Fourier technique. Using such positions as a guide, labor for interpreting electron density maps is much reduced. Here, we report on one example of the structure determination using a selenomethionyl protein as one of the heavy atom derivatives, and give results of the analysis related to the use of selenomethionyl proteins in protein crystallography.
Recent development of softwares for protein crystallography has achieved the recent quick advances of this field. It has enabled us to solve the structures of larger and more complicated proteins and protein complexes in very short period. Especially, electron density modification technology based on real-space restraint has been a very powerful tool for both phase improvement and phase extension, and it has given very expectable effect on an even electron-density map calculated from poor initial empirical phase information. Also recent progress of graphics workstations have produced a new world of protein crystallography. A venous kinds of graphics softwares for model display and building have been developed. Here we introduce recent new technology for improvement of initial empirical phases, and newly developed software for three-dimensional graphics workstations.
X-ray protein crystallography has been extending the target for its application. Membrane proteins and macromolecular super-complexes play important roles in biological systems, but there exist particular problems that should be overcome for their crystallographic analysis. To date appreciable number of membrane proteins and macromolecular super-complexes have been crystallized and the three-dimensional structures for some of them have been successfully determined. Here, the authors describe the problem and progress of their X-ray crystallographic analysis.
We have collected a series of single-exposure Laue diffraction data from trigonal crystals of bovine pancreatic ribonuclease A at the Beam Line 18B in the Photon Factory synchrotron radiation source. Omit Fourier difference maps clearly showed the sulfate anion molecule or the 3'-uridylic acid molecule bound to the catalytic center of ribonuclease A. For time-resolved X-ray crystallographic analyses of biological macromolecules, it is critical to efficiently collect sufficient data for analyzing the target molecule during a single Laue exposure. To accomplish this, it is essential to use a high symmetry crystal and a large size detector.
The crystal structure of cytochrome c oxidase from Paracoccus denitrificans at 2.8Å resolution is described. The crystallization of this membrane protein complex is achieved by co-crystallization with antibody Fv-fragment. The enzyme contains four subunits. Subunit I contains twelve tranmembrane helices and binds heme a and the heme a3-copper B binuclear center where molecular oxygen is reduced to water. Two proton transfer pathways, one for protons consumed in water formation and one for‘proton pumping’could be identified. A possible mechanism for proton pumping is discussed.
Cytochrome c oxidase is a integral membrane protein producing energy in respiratory chain. The enzyme catalyzes reduction of oxygen to water coupled with proton transfer. Structure determination of metal centers of the enzyme having a key role in its function has been a central subject in the field of bio-energetics since discovery of the enzyme. Structures of metal centers, heme a, heme a3, CuA, CUB, Zn and Mg, of bovine heart cytochrome c oxidase were determined by X-ray crystal structure analysis at 2.8Å resolution.
Structure of macrophage migration inhibitory factor has been determined by the method of multiwavelength anomalous diffraction (MAD) with the use of synchrotron data from a crystal of the selenomethionyl protein. The protein contains three methionines in a single polypeptide chain of 114 amino residues. In the analysis, we prepared a series of selenomethionyl proteins by site-directed mutagenesis. The structure was solved using the crystal which contains only one methionine (therefore one Se) per single polypeptide chain.
Aminoacyl-tRNA synthetases (aaRS's) play key roles in the correct protein biosynthesis through strict recognition/ligation of the cognate amino acid and tRNA. Twenty aaRS's were evolutionally devided into two classes (class I and class II) based on the architecture of the ATP-binding domain. Our recent result on the crystal structure of class-I aaRS (glutamyl-tRNA synthetase) delineates that aaRS's accomplish their strict recognition of the tRNA through a combination of insertions and deletions of modular structures in the class/subclass-defining domains and a total exchange of the non-conserved anticodon-binding domains. In contrast, structure of ternary complex of aaRS ·ATP ·amino-acid suggests that the strict amino-acid recognition is accomplished through evolutionary accumulation of mutation of the amino acid residues in the amino-acid binding site.
The crystal structure of vitelline membrane outer layer protein I (VMO-I), which was isolated from the outer layer of the vitelline membrane of hen's eggs, has been determined by X-ray analysis. VMO-I is composed of three homologous structures, each containing a β-sheet forming Greek key motif, which are in accordance with the three repeats in the sequence. These three homologous structures are related by a pseudo three-fold symmetry. This new folding motif, recently designated as the β-prism, has also been observed in the second domain of δ-endotoxin. Thus, VMO-I and δ-endotoxin constitute a new family with a novel folding motif. The VMO-I molecule has a groove-like cavity. This region contains invariant acidic residues in the three repeats. VMO-I is assumed to be an oligosaccharide binding protein like lysozyme, although details of its catalytic function remain to be solved. A docking model of an oligosaccharide to VMO-I has been constructed. This model shows that the cavity has a sufficient space to bind roughly pentamer saccharides. The structural features strongly suggest that the negatively-charged cavity in the top region of the protein may be involved in an unknown catalytic reaction.
The structure of NADH-cytochrome bs reductase has been determined and refined at 2.1 Å resolution. The molecular structure reveals its two domain nature, the FAD binding domain and the NADH domain. Structural difference between the FAD-binding site of this enzyme and that of ferredoxin-NADP+ reductase gives an explanation about the difference of their enzymatic reaction with nucleotides. Three conserved amino acid residues interact significantly with the flavin molecules in four flavin-dependent reductases whose molecular structures have a similar folding pattern in spite of their relative low sequence identity. The electrostatic potentials on the molecular surfaces were compared among these four flavin-dependent reductases and their electron-transfer partners.
SPring-8 is a third-generation synchrotron radiation facility under construction by the JAERI-RIKEN SPring-8 Project Team. Excellent advantages of its undulator sources for protein crystallography are mentioned, and the design concept of the Bio-Crystallography (MIR-OAs) beamline is expressed. This is a public beamline with the undulator source for routine analyses of macromolecular crystallography by the multiple isomorphous replacement phasing with optimized anomalous scattering.
The NMR spectroscopy has been utilized widely for a elucidation of the structural change of a protein caused by the change of pH, ionic strength, temperature, and ligand concentration in solution. The X-ray was less utilized for these study excutable easily in solution, but is utilized much for the structural determination of a protein. Such difference has ever lead to the situation that the NMR relied on the structure solved by X-ray and the X-ray argued its struture in reference to the conformational change elucidated by NMR. However, recent developments of NMR spectroscopy made it possible to determine the three-dimensional structure, and the X-ray techniques has also been developped to clarify the structural change of a protein. This review compares the recent development of these two techniques, and will discuss about the future collaborating interaction between NMR and X-ray.
Elecrton microscopy provides a powerfull means for structural analysis of membrane proteins. Today, using electron microscopy, new exciting structures are getting solved. Structures of some membrane proteins are determined at atomic resolution and some are imaged in the transient states.
The structure-function relationship of proteins is dominated by the behavior of hydrogen atoms. Neutron diffraction provides an experimental method of directly locating hydrogen atoms. We have constructed the diffractometer (BIX) dedicated for the neutron crystallography in biology at JRR-3M in Japan Atomic Energy Research Institute. The data collection from hen egg white lysozyme (HEW-L) is now under way. We have developed an imaging plate neutron detector (IP-ND) . We have succeeded in obtaining the image of Bragg reflections on the IP-ND by using BIX. The neutron pseudo-Laue method equipped with the IP-ND's has provided us complete diffraction data sets of HEW-L for only 10 days machine time.
The purpose of this review is to describe what is deduced about the source of the catalytic power for enzymes, using as guides the crystal structures of the enzymes complexed with transition-state analogs. We start with an overview of transition-state theory and its application to enzyme-catalyzed reactions. We proceed to consideration of the transition-state analogs as probes for the interactions that occur at enzyme active sites. Finally, we describe the case study in an enzyme, glutathione synthetase, and consider a practical approach to enzyme mechanisms using transition-state analogs.
Protein crystallograpy is now a central tool in the drug design process. Using this technique, we can determine atomic resolution structutres for many physiologically important proteins, both in their native state and in complex with a ligand. These data can assist both lead discovery and lead optimization, but at the current time most successful examples have involved the latter, Specific knowledge of the binding site obtained from these structures, enhances the lead optimization process by reducing the number of compounds to be tested. This paper describes the various technologies which are required to make a rational approach successful.
The number of entries saved in the Protein Data Bank in Brookhaven National Laboratory is rapidly increasing, because of the recent development in protein crystallography and NMR spectroscopy. This produce the new field of biology, namely structural biology, and it will combine with the genome science to result information biology. The research fields newly developed utilizing the database are reviewed.
In 1995, we began a cooperative research project for protein crystal structure analysis among industry, public research institutes and universities at the Tsukuba Advanced Research Alliance (TARA) in the University of Tsukuba. This is called the TARA Sakabe Research Project and includes 92 researchers from 13 universities, 4 public institutes and 16 industries as well as 6 foreign researchers. There are 9 research sub-projects in this project. It is my sincere hope that this project will not be limited to X-ray analysis but rather include other tecniques such as NMR, EM, small angle scattering, solution scattering and so on. This would mean that the Sakabe research project could be develop into a structural biology center for a broad range of research related to structure studies. The article written below, describes my personal and tentative plan for the structural biology center.