Crystallization of a scRIP—gelonin isolated from plant seeds Gelonium multiforum

Single crystals of the protein gelonin isolated from the seeds of Gelonium multiforum have been grown at room temperature by vapor diffusion method. The crystals are monclinic with a = 49.4 Å, b = 44.9 Å, c = 137.4 Å, and β = 98.3°. The space group is P2₁, with two molecules in the asymmetric unit which are related by a noncrystallographic 2-fold axis along ψ =13° and ? =88°. The crystals diffract X-rays to high resolution, making it possible to obtain an accurate structure of this single chain ribosome inactivating protein. © 1994 Wiley-Liss, Inc.     

Using cryoEM Reconstruction and Phase Extension to Determine Crystal Structure of Bacteriophage ϕ6 Major Capsid Protein

Bacteriophage ?6 is a double-stranded RNA virus that has been extensively studied as a model organism. Here we describe structure determination of ?6 major capsid protein P1. The protein crystallized in base centered orthorhombic space group C222₁. Matthews’s coefficient indicated that the crystals contain from four to seven P1 subunits in the crystallographic asymmetric unit. The self-rotation function had shown presence of fivefold axes of non-crystallographic symmetry in the crystals. Thus, electron density map corresponding to a P1 pentamer was excised from a previously determined cryoEM reconstruction of the ?6 procapsid at 7 Å resolution and used as a model for molecular replacement. The phases for reflections at higher than 7 Å resolution were obtained by phase extension employing the fivefold non-crystallographic symmetry present in the crystal. The averaged 3.6 Å-resolution electron density map was of sufficient quality to allow model building.     

Characterization of the periplasmic flagellum proteins of Leptospira interrogans.

The structure and composition of periplasmic flagella (PF) from Leptospira interrogans serovar pomona type kennewicki were characterized. Electron microscopic observations showed that leptospiral PF were complex structures composed of an 11.3-nm-diameter core surrounded by two sheath layers with 21.5- and 42-nm diameters. Two-dimensional gel electrophoresis of isolated PF showed the presence of seven different proteins ranging in mass from 31.5 to 36 kDa. Rabbit polyclonal and mouse monoclonal antibodies against PF proteins were prepared and were used to localize specific proteins to portions of the PF structure by immunoelectron microscopy. A 34-kDa protein was associated with the 11.3-nm-diameter core filament, while a 36-kDa protein was associated with a PF sheath (21.5-nm-diameter filament). The amino termini of the 34- and 35.5-kDa proteins were homologous to PF core proteins of other spirochetes. The experimental data suggested that L. interrogans PF contains 2 proteins (34 and 35.5 kDa) in the PF core.     

Genetic analysis of spirochete flagellin proteins and their involvement in motility, filament assembly, and flagellar morphology

The filaments of spirochete periplasmic flagella (PFs) have a unique structure and protein composition. In most spirochetes, the PFs consist of a core of at least three related proteins (FlaB1, FlaB2, and FlaB3) and a sheath of FlaA protein. The functions of these filament proteins remain unknown. In this study, we used a multidisciplinary approach to examine the role of these proteins in determining the composition, shape, and stiffness of the PFs and how these proteins impact motility by using the spirochete Brachyspira (formerly Treponema, Serpulina) hyodysenteriae as a genetic model. A series of double mutants lacking combinations of these PF proteins was constructed and analyzed. The results show the following. First, the diameters of PFs are primarily determined by the sheath protein FlaA, and that FlaA can form a sheath in the absence of an intact PF core. Although the sheath is important to the PF structure and motility, it is not essential. Second, the three core proteins play unequal roles in determining PF structure and swimming speed. The functions of the core proteins FlaB1 and FlaB2 overlap such that either one of these proteins is essential for the spirochete to maintain the intact PF structure and for cell motility. Finally, linear elasticity theory indicates that flagellar stiffness directly affects the spirochete's swimming speed.     

Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli

Crystals of maltoporin (the bacteriophage lambda receptor of Escherichia coli) that diffract X-rays to 3 A resolution can be grown reproducibly. Maltoporin is an integral membrane protein, which forms a channel in the E. coli outer membrane that specifically facilitates the diffusion of maltose and maltodextrins. The crystals have a rhombic prismatic habit and belong to the orthorhombic space group C222(1) with unit cell dimensions a = 130 A, b = 213 A and c = 216 A. X-ray structure determination is underway.     

The three-dimensional structure of bovine platelet factor 4 at 3.0-A resolution

Platelet factor 4 (PF4), which is released by platelets during coagulation, binds very tightly to negatively charged oligosaccharides such as heparin. To date, six other proteins are known that are homologous in sequence with PF4 but have quite different functions. The structure of a tetramer of bovine PF4 complexed with one Ni(CN)4(2-) molecule has been determined at 3.0 A resolution and refined to an R factor of 0.28. The current model contains residues 24-85, no solvent, and one overall temperature factor. Residues 1-13, which carried an oligosaccharide chain, were removed with elastase to induce crystallization; residues 14-23 and presumably 86-88 are disordered in the electron density map. Because no heavy atom derivative was isomorphous with the native crystals, the complex of PF4 with one Ni(CN)4(2-) molecule was solved using a single, highly isomorphous Pt(CN)4(2-) derivative and the iterative, single isomorphous replacement method. The secondary structure of the PF4 subunit, from amino- to carboxyl-terminal end, consists of an extended loop, three strands of antiparallel beta-sheet arranged in a Greek key, and one alpha-helix. The tetramer contains two extended, six-stranded beta-sheets, each formed by two subunits, which are arranged back-to-back to form a "beta-bilayer" structure with two buried salt bridges sandwiched in the middle. The carboxyl-terminal alpha-helices, which contain lysine residues that are thought to be intimately involved in binding heparin, are arranged as antiparallel pairs on the surface of each extended beta-sheet.     

Quantitative Mapping of Protein Structure by Hydroxyl Radical Footprinting-Mediated Structural Mass Spectrometry: A Protection Factor?Analysis

Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.     

The interior of a whole and unmodified biological object--the zymogen granule--viewed with a high-resolution X-ray microscope

We report the ability of focused soft X-rays to visualize at spatial resolution well beyond that of the optical microscope (less than 100 nm) the interior of a small, whole biological object without fixation, staining, dehydration or sectioning. Quantitative estimation of its protein content with unique femtogram sensitivity is also reported. The present results represent a significant step towards the goals of natural imaging and chemical mapping of biological structures with soft X-rays.     

Crystal structure of Pyrococcus furiosus PF2050, a member of the DUF2666 protein family

Pyrococcus furiosus PF2050 is an uncharacterized putative protein that contains two DUF2666 domains. Functional and structural studies of PF2050 have not previously been performed. In this study, we determined the crystal structure of PF2050. The structure of PF2050 showed that the two DUF2666 domains interact tightly, forming a globular structure. Each DUF2666 domain comprises an antiparallel β-sheet and an α-helical bundle. One side of the PF2050 structure has a positively charged basic cleft, which may have a DNA-binding function. Furthermore, we confirmed that PF2050 interacts with circular and linear dsDNA.     

The Armadillo repeat protein PF16 is essential for flagellar structure and function in Plasmodium male gametes

Malaria, caused by the apicomplexan parasite Plasmodium, threatens 40% of the world's population. Transmission between vertebrate and insect hosts depends on the sexual stages of the life-cycle. The male gamete of Plasmodium parasite is the only developmental stage that possesses a flagellum. Very little is known about the identity or function of proteins in the parasite's flagellar biology. Here, we characterise a Plasmodium PF16 homologue using reverse genetics in the mouse malaria parasite Plasmodium berghei. PF16 is a conserved Armadillo-repeat protein that regulates flagellar structure and motility in organisms as diverse as green algae and mice. We show that P. berghei PF16 is expressed in the male gamete flagellum, where it plays a crucial role maintaining the correct microtubule structure in the central apparatus of the axoneme as studied by electron microscopy. Disruption of the PF16 gene results in abnormal flagellar movement and reduced fertility, but does not lead to complete sterility, unlike pf16 mutations in other organisms. Using homology modelling, bioinformatics analysis and complementation studies in Chlamydomonas, we show that some regions of the PF16 protein are highly conserved across all eukaryotes, whereas other regions may have species-specific functions. PF16 is the first ARM-repeat protein characterised in the malaria parasite genus Plasmodium and this study opens up a novel model for analysis of Plasmodium flagellar biology that may provide unique insights into an ancient organelle and suggest novel intervention strategies to control the malaria parasite.     

Structural characterization of penicillin-binding protein-related factor A (PrfA) from Bacillus species

The prfA genes of Bacillus stearothermophilus and Bacillus subtilis are in an operon downstream of the ponA gene encoding penicillin-binding protein 1 (PBP1), a major enzyme involved in peptidoglycan synthesis. The specific function of the 23- to 24-kDa PrfA protein is unknown but this protein plays some role in nucleoid segregation and the functions of PrfA and PBP1 are interrelated. We overexpressed B. stearothermophilus and B. subtilis PrfA in Escherichia coli and purified the proteins to homogeneity by cation exchange and gel filtration chromatography. The protein is a monomer in solution, and circular dichroism spectroscopy revealed an abundance of beta-sheet secondary structure. Crystals of B. stearothermophilus PrfA were also obtained and diffracted X-rays to 1.8 A resolution.     

Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica

The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5-30°C with maximal activity at 15-20°C. The esterase activity of OLEI01171 was stimulated 3-8-fold by the addition of chloride or several other anions (0.1-1.0?M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures of OLEI01171 were solved at 1.75 and 2.1?? resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148. Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35-45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.     

Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.     

Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.     

X-Ray-Radiation-Induced Cooperative Atomic Movements in Protein

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is.We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to ~ 100 K and diffracting to 1 ?. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.     

The gpQ portal protein of bacteriophage P2 forms dodecameric connectors in crystals

Double-stranded bacteriophages code for a protein called a connector or portal protein that serves as the entry and exit portal for DNA during genome packaging and ejection, as well as the connection point between heads and tails, and possibly as a nucleator for capsid assembly. The gpQ connector protein from bacteriophage P2 has been overexpressed in Escherichia coli and purified by sucrose gradient centrifugation. Negative stain electron microscopy and image analysis revealed a 135 A diameter dodecameric ring structure with a central 25 A hole. The connector showed a strong propensity to aggregate at low ionic strength and would form microcrystalline structures in solution. Consequently, the connectors were crystallized by hanging-drop vapor diffusion against low ionic strength buffer. Two crystal forms were observed: a P4(1)22 form with unit cell parameters a=b=96.33 A and c=454.42 A that diffracted X-rays to 4.5 A resolution and an I222 crystal form with a=168.86 A, b=171.88 A and c=168.68 A that diffracted to 4.1A resolution. Self-rotation functions confirmed the presence of 12-fold symmetry in the crystals.     

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.     

Qualitative and Quantitative analysis of 3D predicted arachidonate 15-lipoxygenase-B (15-LOX-2) from Homo sapiens

15-Lipoxygenase-2 protein has been reported to play an important role in normal development of prostate, lung, skin, and cornea tissues. It behaves as a suppressor of prostate cancer development by restricting cell cycle progression and implicating a possible protective role against tumor formation. On the basis of the above report, we selected 15-LOX-2 protein to study the structural classification and functional relationship with associated protein network at computational level. Sequence alignment and protein functional study shows that it contains a highly conserved LOX motif. PLAT domain with PF01477 and LH2 domain with PF00305 were successfully observed. Arachidonate 5-lipoxygenase (PDB ID: 3O8Y) was selected as a template with 42% identity. 3D structure was successfully predicted and verified. Qualitative analysis suggests that the predicted model was reliable and stable with best quality. Quantitative study shows that the model contained expected volume and area with best resolution. Predicted and best evaluated model has been successfully deposited to PMDB database with PMDB ID PM0078035. Active site identification revealed GLU(369), ALA(370), LEU(371), THR(372), HIS(373), LEU(374), HIS(376), SER(377), HIS(378), THR(385), LEU(389), HIS(394), PHE(399), LYS(400), LEU(401), ILE(403) and PRO(404) residues may play a major role during protein-protein, protein-drug and protein-cofactor interactions. STRING database result indicated that IL (4), GPX (2 and 4), PPARG, PTGS (1 and 2), CYP (2J2, 2C8, 4A11 and 2B6), PLA (2G2A, 2G4A, 2G1B and 2G6) and A LOX (5, 15, 12 and 12B) members from their respective gene families have network based functional association with 15-LOX-2.