Crystal structure analysis of recombinant human uteroglobin and molecular modeling of ligand binding.

Uteroglobin, a steroid-inducible, cytokine-like, secreted protein with immunomodulatory properties, has been reported to bind progesterone, polychlorinated biphenyls (PCB), and retinol. Structural studies may delineate whether binding of ligands is a likely physiological function of human uteroglobin (hUG). We report a refined crystal structure of uncomplexed recombinant hUG (rhUG) at 2.5-A resolution and the results of our molecular modeling studies of ligand binding to the central hydrophobic cavity of rhUG. The crystal structure of rhUG is very similar to that of reported crystal structures of uteroglobins. Using molecular modeling techniques, the three ligands--PCB, progesterone, and retinol--were docked into the hydrophobic cavity of the dimer structure of rhUG. We undocked the progesterone ligand by pulling the ligand from the cavity into the solvent. From our modeling and undocking studies of progesterone, it is clear that these types of hydrophobic ligands could slip into the cavity between helix-3 and helix-3' of the dimer instead of between helix-1 and helix-4 of the monomer, as proposed earlier. Our results suggest that at least one of the physiological functions of UG is to bind to hydrophobic ligands, such as progesterone and retinol.

Occluded molecular surface: analysis of protein packing.

We describe a novel method to calculate the packing interactions in protein structural models. The method calculates the interatomic occluded surface areas for each atom in the protein model. The identification of, and degree of interaction with, neighboring atoms is accomplished by extending surface normals from a dot surface of each atom to the point of intersection with neighboring atoms. The combined occluded and non-occluded surface areas may be normalized for the amino acid composition of the protein providing a single parameter, the normalized protein surface ratio, which is diagnostic for native-like structures. Individual residues in the model which are in infrequent occluded surface environments may be identified. The method provides a means to explicitly describe packing densities and packing environments of individual atoms in a protein model. Finally, the method allows estimation of the complementarity between any interacting molecules, for example a ligand binding to a receptor.

Rapid purification of recombinant green fluorescent protein using the hydrophobic properties of an HPLC size-exclusion column.

The green fluorescent protein (GFP) of the jelly fish Aequoria victoria was cloned into an Escherichia coli cell line that is a methionine auxotroph. The recombinant GFP (rGFP) was isolated from the cells and purified using a simple procedure consisting of only two chromatographic steps: size-exclusion chromatography and ion-exchange HPLC. Due to the hydrophobic nature of the protein, the surface characteristics of the HPLC size column, and the high initial salt concentration, the rGFP sticks to the size column and is eluted by reducing the salt concentration. Due to this unique behavior the purification procedure can readily be scaled to handle larger quantities of rGFP.

Crystallization and characterization of the recombinant human Clara cell 10-kDa protein.

Crystals of recombinant human Clara cell 10-kDa protein were grown both from ammonium sulfate and polyethylene glycol (PEG) solutions. Crystals grown from ammonium sulfate solution have been characterized by X-ray diffraction studies as monoclinic with the space group C2 and lattice constants a = 69.2 A, b = 83.0 A, c = 58.3 A, and beta = 99.7 degrees. The monoclinic crystals diffract to beyond 2.5 A. Some of the crystals grown from PEG were of a similar habit to those grown from ammonium sulfate, but others were triclinic with the space group P1 and cell constants a = 40.3 A, b = 46.3 A, c = 51.3 A, alpha = 117.7 degrees, beta = 102.3 degrees, and gamma = 71.4 degrees. These crystals diffract to beyond 3.2 A.

Crystal structure of deltakephalin: a delta-selective opioid peptide with a novel beta-bend-like conformation.

The solid-state structure of deltakephalin (Tyr-DThr-Gly-Phe-Leu-Thr) has been determined by single-crystal X-ray diffraction. Deltakephalin (DTLET) is a synthetic opioid peptide which differs from enkephalin in that a D-Thr has been substituted for Gly2 and a sixth residue, L-Thr, has been added. Clear colorless plates obtained using vapor diffusion and macro-seeding crystallization techniques were monoclinic; space group C2 with a = 27.389(5), b = 9.205(2), c = 16.788(2) A, beta = 98.87(2) degrees and V = 4181.4(14) A3. The asymmetric unit contained one molecule of DTLET and six molecules of water, giving a calculated density of 1.28 g cm-3. The crystal structure revealed that DTLET has a pseudo type I' beta-bend which is stabilized by an intramolecular side-chain to backbone hydrogen bond. This is the first reported observation of a pseudo beta-bend conformation in a solid-state structure of an enkephalin analog.

Observation of growth steps, spiral dislocations and molecular packing on the surface of lysozyme crystals with the atomic force microscope.

The (110) faces of lysozyme crystals in their mother liquor have been investigated using an atomic force microscope (AFM) in height mode. Crystal growth and dissolution steps, as well as simultaneous growth and dissolution in pits, have been observed. Screw dislocations were also observed but the fine structure has not yet been investigated. Images that may possess molecular resolution were obtained and compared with theoretical images based on the crystallographic structure and the effects of arbitrary tip profiles. Crystallographic periodicities of 38 and 112 A were observed. A recurring feature is a centered periodic array of minima that may be associated with one of the two nearly planar sheets of molecules present in the crystal that are parallel to the (110) faces.

Comparison of organophosphorous acid anhydrolases from different species using monoclonal antibodies.

1. Monoclonal antibodies were raised against squid hepatopancreas organophosphorous acid (OPA) anhydrolase (EC 3.1.8.2) and were used to study structural similarities with OPA anhydrolases isolated from different sources. 2. Common epitopes were identified in OPA anhydrolases with diverse origins, and with different substrate specificities. 3. Epitopes unique to the squid hepatopancreas OPA anhydrolase were identified; optic ganglion and hepatopancreas contain different enzymes which can be distinguished by their epitopes.

A partial primary structure of squid hepatopancreas organophosphorus acid anhydrolase.

Earlier studies of OPA anhydrolase from the squid, Loligo pealei, report that the enzyme has a molecular weight near 26 kDa, despite the common observation that SDS-PAGE experiments do not support this conclusion. Recent results from protein sequencing and cloning experiments now suggest that the enzyme found in squid hepatopancreas has a molecular weight of about 42 kDa. The enzyme easily degrades into two fragments of 16 kDa and approximately 26 kDa. N-terminal sequence analyses of the intact enzyme and the 16 kDa fragment blotted from an SDS gel and sequenced from the blot have shown conclusively that the intact 42 kDa protein has a blocked N-terminus. Sequence data obtained previously are from the N-terminal portion of the 16 kDa fragment. Additional support for this interpretation has been obtained from PCR analysis of L. pealei mRNA and cDNA. The partial (30 residue) sequence presented here reveals no indication of similarity to any other OPA anhydrolase or aryldialkylphosphatase (EC 3.1.8.1.).

Preparation and initial characterization of crystals of the photoprotein aequorin from Aequorea victoria.

Crystals of recombinant aequorin, the photoprotein from the jellyfish Aequorea victoria, have been grown from solutions containing sodium phosphate. The crystals grow as thin plates which diffract to beyond 2.2 A resolution. The crystals are orthorhombic, space group P2(1)2(1)2(1); the axes are a = 89.1(1), b = 88.4(1), and c = 52.7(1) A. The asymmetric unit contains two molecules. Crystals exposed to calcium ion solutions emit a steady glow and slowly deteriorate, confirming that the crystals consist of a charged, competent photoprotein. This represents the first successful preparation of single crystals of a photoprotein suitable for diffraction analysis.

Crepe-ribbon representation for protein structures: comparison of phospholipases A2.

We describe a method to generate a novel representation for protein structures called "crepe ribbons." In our representation, each piece of the ribbon was constructed using the coordinates of the backbone atoms of each individual residue. Using the internal geometries of each residue and a helix-generating algorithm, a local origin and the direction cosines of a local orthogonal system were obtained. The locus of the local origins represents the folding of the polypeptide. A color-coded origin-origin distance plot similar to that of C alpha-C alpha distance plot was generated. This plot may be used to visually compare and contrast two structures. We identified linear regions in the distribution of the local origins and assigned a secondary structure description. Parameters describing the interrelation between various secondary structure segments were calculated. We have illustrated our crepe-ribbon representation by comparing two phospholipase A2 structures in the Brookhaven National Laboratory Protein Data Bank.

X-ray diffraction and time-resolved fluorescence analyses of Aequorea green fluorescent protein crystals.

The energy transfer protein, green fluorescent protein, from the hydromedusan jellyfish Aequorea victoria has been crystallized in two morphologies suitable for x-ray diffraction analysis. Hexagonal plates have been obtained in the P6122 or P6522 space group with a = b = 77.5, c = 370 A, and no more than three molecules per asymmetric unit. Monoclinic parallel-epipeds have been obtained in the C2 space group with a = 93.3, b = 66.5, c = 45.5 A, beta = 108 degrees, and one molecule per asymmetric unit. The monoclinic form is better suited for use in a structure determination, and a data set was collected from the native crystal. Time-resolved fluorescence measurements of large single crystals are possible due to the unique, covalently bound chromophore present in this molecule. Fluorescence emission spectra of Aequorea green fluorescent protein in solution and from either the hexagonal or monoclinic single crystal show similar profiles suggesting that the conformations of protein in solution and in the crystal are similar. Multifrequency phase fluorimetric data obtained from a single crystal were best fit by a single fluorescence lifetime very close to that exhibited by the protein in solution. The complementary structural data obtained from fluorescence spectroscopy and x-ray diffraction crystallography will aid in the elucidation of this novel protein's structure-function relationship.

The 2.5 A crystal structure of a dimeric phospholipase A2 from the venom of Crotalus atrox.

The crystal structure of the dimeric (alpha 2) phospholipase A2 from Crotalus atrox has been determined by multiple isomorphous replacement to 2.5 A resolution. A skeletal model was fit to the electron density, and the stereochemistry of the backbone was idealized. The dimeric molecule is a well defined oblate ellipsoid composed of two covalently identical subunits related by a local dyad axis which is essentially "exact" except for deviations at the periphery induced by ionic lattice contacts with neighboring dimers. As expected, the basic architecture of the individual protomers is similar to the structure of the homologous monomeric bovine enzyme (Dijkstra, B. W., Drenth, J., Kalk, K., and Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60). The intramolecular contact surface between the protomers is extensive and involves the catalytic and calcium-binding sites. Access to an internal cavity formed by the enclosed and abutting active center regions is quite restricted. The putative interfacial recognition surfaces of each protomer are exposed to the solvent but are on opposing surfaces of the ellipsoid, suggesting that both of these regions cannot interact with the same membrane surface simultaneously unless the membrane is distorted from planarity and/or the dimer is significantly modified.

Pseudosymmetry in the structure of myohemerythrin.

The three-dimensional structure of the protein myohemerythrin from retractor muscles of the sipunculan worn Themiste zostericola has been explored for the existance of approximately symmetry operators that locally interrelate portions of the molecule. First, the electron denisty distribution at 5.5 A resolution was examined. A local 2-fold axis that transposes the C-D helix pair into the A-B helix pair was found and refined by the method of least squares. The match in electron densities for a pure 2-fold rotation had a correlation coefficient of 0.56. Next, a comprehensive search was made for rotational symmetry in the Patterson function of an isolated molecule. The rotation function based on data to 6 A spacings showed a major peak, 72% of the self-peak height, that confirmed the result from electron density correlations. In addition, a pattern of lower level peaks revealed approximate point group symmetry as high as D4. Finally, the amino acid sequence has been inspected for evidence of a repeated structure. The level of amino acid identities between positions in the A-B and C-D helix pairs is 28%. Several factors are discussed to suggest that this homology, although low, is nonetheless significant.

Tertiary structure of myohemerythrin at low resolution.

X-ray diffraction studies have produced a low resolution image and also located the iron atoms of a monomeric hemerythrin from muscles of a sipunculan worm. These results reveal the course of the polypeptide chain and some details of the active center.