Structure of the Kunitz-type soybean trypsin inhibitor (STI): implication for the interactions between members of the STI family and tissue-plasminogen activator.

The Kunitz-type soybean trypsin inhibitor (STI) has played a key role in the early study of proteinases, having been used as the main substrate in the biochemical and kinetic work that led to the definition of the standard mechanism of action of proteinase inhibitors. A partial structure of STI complexed with porcine trypsin has previously been reported, in which the first 93 residues of the inhibitor, including the region of contact with trypsin, were relatively well defined, whereas for the remaining part of the peptide chain only some Calpha atoms were located. The structure of the inhibitor in its free form has now been determined by molecular replacement to 2.5 A, using the coordinates of the homologous Erythrina trypsin inhibitor as a search model. When the refined atomic coordinates of STI are compared with the partial model previously available, the conformation of the reactive-site loop and its position with respect to the main body of the molecule does not change when the inhibitor interacts with trypsin. There are instead, despite the high similarity in the overall tertiary structure, significant differences between STI and Erythrina trypsin inhibitor (ETI) in the region which is in contact with the enzyme in the STI:trypsin crystal structure. Some of these differences can explain the unique specificity of ETI and its ability to inhibit the fibrinolytic enzyme tissue-type plasminogen activator.

Crystallization and preliminary diffraction studies of firefly luciferase from Photinus pyralis.

Firefly luciferase is a 62 kDa molecular weight enzyme which catalyzes a light-emitting reaction. Crystals of Photinus pyralis luciferase have been obtained by the microbatch technique, using polyethylene glycol as a precipitating agent. Firefly luciferase crystallizes as long needles which belong to the tetragonal space group P4(1)2(1)2, with cell dimensions a = 119.5, b = 119.5, c = 95.4 A. One molecule is present in the asymmetric unit. Diffracted intensities beyond 2.0 A resolution have been measured from frozen crystals using synchrotron radiation.

Structure of full-length porcine synovial collagenase reveals a C-terminal domain containing a calcium-linked, four-bladed beta-propeller.

BACKGROUND: The collagenases are members of the family of zinc-dependent enzymes known as the matrix metalloproteinases (MMPs). They are the only proteinases that specifically cleave the collagen triple helix, and are important in a large number of physiological and pathological processes. Structures are known for the N-terminal catalytic' domain of collagenases MMP-1 and MMP-8 and of stromelysin (MMP-3). This catalytic domain alone, which comprises about 150 amino acids, has no activity against collagen. A second domain, of 200 amino acids, is homologous to haemopexin, a haem-binding glycoprotein. RESULTS: The crystal structure of full-length MMP-1 at 2.5 A resolution gives an R-factor of 21.7%. Two domains are connected by an exposed proline-rich linker of 17 amino acids, which is probably flexible and has no secondary structure. The catalytic domain resembles those previously observed, and contains three calcium-binding sites. The haemopexin-like domain contains four units of four-stranded antiparallel beta sheet stabilized on its fourfold axis by a cation, which is probably calcium. The domain constitutes a four-bladed beta-propeller structure in which the blades are scarcely twisted. CONCLUSIONS: The exposed linker accounts for the difficulty in purifying full-length collagenase. The C-terminal domain provides a structural model for haemopexin and its homologues. It controls the specificity of MMPs, affecting both substrate and inhibitor binding, although its role remains obscure. These structural results should aid the design of site-specific mutants which will reveal further details of the specificity mechanism.

Control of nucleation of protein crystals.

Control of nucleation may be needed to obtain a reliable supply of large protein crystals, when standard techniques give many small or twinned crystals. Heterogeneous nucleation may be controlled by the use of fine filters, with the elimination of airborne contaminants by working under paraffin oil. The area of contact with the supporting vessel also has an important effect. A heterogenous nucleant for lysozyme (identified earlier) has been shown to be effective for carboxypeptidase G2. Control of homogeneous nucleation (previously demonstrated by dilutions of a nucleating sample after various times of incubation) may also be achieved by incubating a sample at 1 temperature, where nucleation can occur, and changing the temperature to conditions where there is growth but no nucleation.

Maintenance and generalization of behaviours after treatment of persons with traumatic brain injury.

This review of behavioural research literature was conducted to determine if persons who have experienced a traumatic brain injury are able to maintain and generalize behaviours after training, and to identify formal programming strategies that might have been utilized to enhance maintenance and generalization. Studies reviewed included those that employed behavioural procedures to increase adaptive behaviours or reduce maladaptive behaviours and collected maintenance and generalization data. Results of the review indicate that persons who have experienced a traumatic brain injury have experienced successful response maintenance and generalization of community-referenced tasks. It is suggested that impaired memory does not keep persons from maintaining and generalizing such tasks, and that strategies to promote maintenance and generalization need to be included in behavioural programming.

Crystallization and preliminary X-ray diffraction studies of xylose isomerase from Thermoanaerobacterium thermosulfurigenes strain 4B.

Xylose isomerase from the thermophile Thermoanaerobacterium thermosulfurigenes strain 4B has been crystallized by vapour diffusion from Jeffamine ED 4000 as precipitant. The crystal symmetry is P2(1)2(1)2(1), with unit cell dimensions a = 85.6 A, b = 153.5 A and c = 158.5 A. The protein molecular mass and volume of the unit cell is consistent with the presence of a tetramer of the enzyme in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 A resolution.

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2.

The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 x 0.3 x 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16.

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.

Crystal structure of cholesterol oxidase from Brevibacterium sterolicum refined at 1.8 A resolution.

Cholesterol oxidase (3 beta-hydroxysteroid oxidase, EC 1.1.3.6) is an FAD-dependent enzyme that carries out the oxidation and isomerization of steroids with a trans A : B ring junction. The crystal structure of the enzyme from Brevibacterium sterolicum has been determined using the method of isomorphous replacement and refined to 1.8 A resolution. The refined model includes 492 amino acid residues, the FAD prosthetic group and 453 solvent molecules. The crystallographic R-factor is 15.3% for all reflections between 10.0 A and 1.8 A resolution. The structure is made up of two domains: an FAD-binding domain and a steroid-binding domain. The FAD-binding domain consists of three non-continuous segments of sequence, including both the N terminus and the C terminus, and is made up of a six-stranded beta-sheet sandwiched between a four-stranded beta-sheet and three alpha-helices. The overall topology of this domain is very similar to other FAD-binding proteins. The steroid-binding domain consists of two non-continuous segments of sequence and contains a six-stranded antiparallel beta-sheet forming the "roof" of the active-site cavity. This large beta-sheet structure and the connections between the strands are topologically similar to the substrate-binding domain of the FAD-binding protein para-hydroxybenzoate hydroxylase. The active site lies at the interface of the two domains, in a large cavity filled with a well-ordered lattice of 13 solvent molecules. The flavin ring system of FAD lies on the "floor" of the cavity with N-5 of the ring system exposed. The ring system is twisted from a planar conformation by an angle of approximately 17 degrees, allowing hydrogen-bond interactions between the protein and the pyrimidine ring of FAD. The amino acid residues that line the active site are predominantly hydrophobic along the side of the cavity nearest the benzene ring of the flavin ring system, and are more hydrophilic on the opposite side near the pyrimidine ring. The cavity is buried inside the protein molecule, but three hydrophobic loops at the surface of the molecule show relatively high temperature factors, suggesting a flexible region that may form a possible path by which the substrate could enter the cavity. The active-site cavity contains one charged residue, Glu361, for which the side-chain electron density suggests a high degree of mobility for the side-chain. This residue is appropriately positioned to act as the proton acceptor in the proposed mechanism for the isomerization step.

Many crystal forms of human immunodeficiency virus reverse transcriptase.

Many crystal forms of human immunodeficiency virus reverse transcriptase have been obtained by vapour diffusion, microbatch and microdialysis methods. Despite their apparent morphological perfection, no X-ray diffraction has been discernible in most cases with these crystals.

Crystallization and preliminary diffraction studies of Erythrina trypsin inhibitor.

Crystals of an inhibitor of trypsin and tissue plasminogen activator from seeds of the legume Erythrina caffra have been obtained by vapour diffusion. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22) with cell parameters 73.4 A and 143.0 A. There is one molecule in the asymmetric unit. The crystals diffract to beyond 2.5 A resolution.

Crystallization and preliminary X-ray analysis of porcine synovial collagenase.

Crystals of porcine synovial collagenase suitable for an X-ray structure analysis have been obtained. The crystals belong to space group I4, with unit cell dimensions a = b = 160.0 A, c = 53.1 A, with one molecule in the asymmetric unit. Diffraction extends beyond 3 A perpendicular to the c axis but along the 4-fold axis, the intensities are measurable only to 4 A.