With the sequencing of the genomes of various species, attention has turned to the structure, properties, and functional activities of proteins. However, rapid progress in the area of proteomics is premised on the availability of sufficient amounts of a large number of proteins. Here we described a novel cell-free system from wheat embryos for the high-throughput screening/ synthesis of gene products. Our system should open up many possibilities in the post-genome era.
A basis for protein crystal preparation for high resolution structure determination is provided for vertebrate cytochrome c. Crystals are shown to grow in the presence of ammonium sulfate and sodium nitrate for tuna, horse and bovine cytochrome c. All the crystals obtained diffracted X-ray to a resolution better than 2.0 Å. X-ray crystallography revealed that a nitrate ion combines two protein molecules near the C-terminal, thus stabilizing the terminal region. A combined use of two salts for crystallization is considered to be a reasonable and rationalized way of growing high quality crystals for this protein.
The crystallographic phase problem had not been routinely solved in protein crystallography. Nowadays, MAD method is widely used for crystallographic determination of unknown protein structures, since the scope of MAD application has been extended by the use of synchrotron radiation facilities, protein expression techniques, and computational development. The method has paved the way to high-throughput structure determination of protein crystals and introduced to structural genomics study. We describe here a brief summary of MAD method and its experimental.
In the eukaryotic cells, the gene expressions are regulated by multiple transcriptional regulatory factors bound to the promoter regions of the target genes. The DNA-binding activity and trans-activating capacity of these transcription factors are precisely controlled via synergistic intermolecular interactions. Based on the structural analyses using X-ray crystallography, atomic force microscopy and temperature-scanning spectroscopy and the functional analyses using various molecular interaction experiments (including GST pull down assay, gel shift assay, calorimetry and surface plasmon resonance experiments) and trans-activation assays, the underlying molecular mechanisms of the transcriptional regulations in the hematopoietic system and their deregulated states in the leukemic cells are presented.
Many eukaryiotic proteins are glycosylated after translation to become mature proteins. Efficient sorting of such glycosylated proteins is essential for the cell's function and is achieved by vesicle transport. Mannose 6-phosphate (M6P) modification of lysosomal proteins is recognized by a M6P receptor (MPR) which incorporates the lysosomal proteins to vesicles. Recently GGA proteins have been identified and later verified as a new family of adapter proteins distinct from well known AP-1 to 4 complexes. They are responsible for vesicle transport of glycosylated human proteins from the Golgi apparatus to early endosomesl lysosomes. We have determined the crystal structures of the VHS domain of human GGA 1 and the ear domain of human γ1 adaptin. Combined with biochemical and cell-biological data, these structures reveal the recognition mechanisms of the acidic dileucine motif signal of mannose-6-phophate receptor by the VHS domain of human GGA protein and the interaction between the ear domain of γ1 adaptin of the AP-1 complex and γ-synergin and Rabaptin-5. They constitute part of a rapidly growing ensemble of structures of transport proteins, where protein-protein interactions, as elucidated by X-ray structural analyses, play critical roles.
The actin filament is a flexible and easy-to- (dis-) assemble structure. In order to understand the mechanism of calcium regulation of muscle contraction, we have recently obtained the crystal structure of troponin, which implied that troponin may regulate the flexibility of actin /tropomyosin filament. The questions to address are, on one hand, how to prepare “mini-actin filaments” suitable for crystallization, and on the other, how to deduce dynamic properties of the actin filament from the static structure.
The bacterial flagellum is a motility apparatus in which a helical filament is driven by a rotary motor. The long helical filament, which acts as a screw, is not a rigid propeller, but switches its helical form upon quick reversal of the motor rotaion. Complementary use of Xray diffraction and electron cryomicroscopy reveals the detail structure of this large complex. We describe the molecular mechanism of polymorphic supercoiling based on the structure.
The ribosome is a ribonucleoprotein complex responsible for protein synthesis. The three-dimensional structures of the ribosome subunits have elucidated the mechanisms of this translation machinery in molecular basis. The relationships between the proteins and RNAs in this ribonucleoprotein complex are discussed.
The sub-atomic structure of the tail-lysozyme complex of bacteriophage T4 has been determined to the resolution of 2.9 Å. For the phase determination, MAD (mufti-wavelength anomalous dispersion) from seleno-methionine-substituted gp27 which had been complexed with unlabeled gp5 (gp = gene product) was utilized. The tail-lysozyme was then localized in the low-resolution structure of the tail baseplate, which revealed the role of the C-terminal β-helix domain as a cell-puncturing device as well as an intra-molecular chaperone to form the trimeric tail-lysozyme complex.
The folding of protein molecules and the maintenance of their biological functions are controlled by molecular chaperons, which are good targets for structural biology of biological machinery. Crystal structures and structural conversion of chaperonin system (groups I and II) are reviewed as a typical example of structural biology of molecular chaperones and a view for further crystallographic studies in this field is discussed.
Calcium pump (Ca2+-ATPase) of muscle sarcoplasmic reticulum is an integral membrane protein of Mr 110 k and a representative member of P-type ATPases involved in the active transport of ions across the membrane using the energy liberated by the hydrolysis of ATP. Crystal structures of this enzyme has now been determined for both calcium bound and unbound states. The structures exhibit a very large scale domain movements between the two states and provide insight into the mechanism of active transport. These pumps appear to work in a similar way to mechanical pumps at an atomic scale.
The living organism is a complex system consisting of numerous biological machineries that are defined as a functional unit responsible for particular biological activities, such as protein synthesis, ATP synthesis, and various kinds of metabolisms. Biological machineries may be classified into two types. The first one refers to stable assembly systems of macromolecules. This kind of machineries may sometimes be crystallized and their whole structures are determined by the X-ray crystallography. The second one does not create such a stable assembly system, but by the concerted acts of the individual macromolecules, the systems play as whole complex biological roles. The development of protein crystallography in recent years makes it possible to determine the overall three-dimensional structures of the biological machinery of both types. Three-dimensional structure determination of biological machineries would open a new era in the field of structural biology.
Two dioxygenases found in the PCB degradation pathway, biphenyl dioxygenase and extradiol type catecholic dioxygenase, play key roles in the pathway. The crystal structures of reaction intermediates of the dioxygenases have revealed the catalytic mechanisms of theses enzymes.
Recent progresses in crystallography on biological macromolecules enable us to solve the atomic structure of the huge macromolecule assemblies. High-brilliance synchrotron radiation and a high-performance area detector are essential for high-quality diffraction data collection from a small-size crystal. Newly developed software for crystal structure determination can be used for quick and accurate structure determinaion. In this paper, we will review the recent progress in crystallography on biological macromolecular assemblies.