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Protein crystallization has gained a new strategic and commercial relevance in the postgenomic era due to its pivotal role in structural genomics. Producing high quality crystals has always been a bottleneck to efficient structure determination, and this problem is becoming increasingly acute. This is especially true for challenging, therapeutically important proteins that typically do not form suitable crystals. The OptiCryst consortium has focused on relieving this bottleneck by making a concerted effort to improve the crystallization techniques usually employed, designing new crystallization tools, and applying such developments to the optimization of target protein crystals. In particular, the focus has been on the novel application of dual polarization interferometry (DPI) to detect suitable nucleation; the application of in situdynamic light scattering (DLS) to monitor and analyze the process of crystallization; the use of UV-fluorescence to differentiate protein crystals from salt; the design of novel nucleants and seeding technologies; and the development of kits for capillary counterdiffusion and crystal growth in gels. The consortium collectively handled 60 new target proteins that had not been crystallized previously. From these, we generated 39 crystals with improved diffraction properties. Fourteen of these 39 were only obtainable using OptiCryst methods. For the remaining 25, OptiCryst methods were used in combination with standard crystallization techniques. Eighteen structures have already been solved (30% success rate), with several more in the pipeline.

Background: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Šresolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. Methods: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Ů The ligand affinity was verified by in-silico docking. Results: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. Conclusion: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs.

The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5’- nucleotidases (5’-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes.

The gene coding for the aminoglycoside adenylyltransferase (aadA6) from a clinical isolate of Pseudomonas aeruginosawas cloned and expressed in Escherichia colistrain BL21(DE3)pLysS. The overexpressed enzyme (AadA6, 281 amino-acid residues) and a carboxy-terminal truncated variant molecule ([1-264]AadA6) were purified to near homogeneity and characterized. Light scattering experiments conducted under low ionic strength supported equilibrium between monomeric and homodimeric arrangements of the enzyme subunits. Circular Dichroism spectropolarimetry indicated a close structural relation to adenylate kinases. Both forms modified covalently the aminoglycosides streptomycin and spectinomycin. The enzyme required at least 5mM MgCl2for normal Michaelis–Menten kinetics. Streptomycin exhibited a strong substrate inhibition effect at 1mM MgCl2. The truncated 17 residues at the C-terminus have little influence on protein folding, whereas they have a positive effect on the enzymic activity and stabilize dimers at high protein concentrations (>100μM). Homology modelling and docking based on known crystal structures yielded models of the central ternary complex of monomeric AadA6 with ATP and streptomycin or spectinomycin.

Infections caused by the methicillin-resistant Staphylococcus aureus (MRSA) are today a major burden in nosocomial disease control. The global trend shows an alarming increase of MRSA infections as well as multi-drug resistance (MDR). The problem is exacerbated by the fact that infections with community-associated (CA) MRSA strains showing increased virulence and fitness add to infections with multi-drug resistant hospital-associated (HA) MRSA. The toxicity of pathogens and limited effectiveness of available treatment have led to high mortality rates and vast expenses caused by prolonged hospitalization and usage of additional antibiotics. Recently approved drugs still have classical targets and upcoming resistance can be expected. In a new approach by targeting co-factor syntheses of bacteria, the drug target and the affected pathways are uncoupled. This novel strategy is based on the thought of a classical pro-drug which has to be metabolized before becoming toxic for the bacterium as a dysfunctional co-factor, named suicide drug. Ideally these metabolizing pathways are solely present in the bacterium and absent in the human host, such as vitamin biosyntheses. This mini-review discusses current ways of MRSA infection treatment using new approaches including suicide drugs targeting co-factor biosyntheses.

Prion diseases comprise a group of fatal neurodegenerative disorders characterized by the autocatalytic conversion of the cellular prion protein PrPCinto the infectious misfolded isoform PrPSc. Increasing evidence supports a specific role of oxidative stress in the onset of pathogenesis. Although the associated molecular mechanisms remain to be elucidated in detail, several studies currently suggest that methionine oxidation already detected in misfolded PrPScdestabilizes the native PrP fold as an early event in the conversion pathway. To obtain more insights about the specific impact of surface-exposed methionine residues on the oxidative-induced conversion of human PrP we designed, produced, and comparatively investigated two new pseudosulfoxidation mutants of human PrP 121–231 that comprises the well-folded C-terminal domain. Applying circular dichroism spectroscopy and dynamic light scattering techniques we showed that pseudosulfoxidation of all surface exposed Met residues formed a monomeric molten globule-like species with striking similarities to misfolding intermediates recently reported by other groups. However, individual pseudosulfoxidation at the polymorphic M129 site did not significantly contribute to the structural destabilization. Further metal-induced oxidation of the partly unfolded pseudosulfoxidation mutant resulted in the formation of an oligomeric state that shares a comparable size and stability with PrP oligomers detected after the application of different other triggers for structural conversion, indicating a generic misfolding pathway of PrP. The obtained results highlight the specific importance of methionine oxidation at surface exposed residues for PrP misfolding, strongly supporting the hypothesis that increased oxidative stress could be one causative event for sporadic prion diseases and other neurodegenerative disorders.

Api SI and Api SII are serine proteases of the honeybee venom containing allergenic determinants. Each protease consists of two structural modules: an N-terminal CUB (Api SI) or a clip domain (Api SII) and a C-terminal serine protease-like (SPL) domain. Both domains are connected with a linker peptide. The knowledge about the structure and function of Api SI and Api SII is limited mainly to their amino acid sequences. We constructed 3-D models of the two proteases using their amino acid sequences and crystallographic coordinates of related proteins. The models of the SPL domains were built using the structure of the prophenoloxidase-activating factor (PPAF)-II as a template. For modelling of the Api SI CUB domain the coordinates of porcine spermadhesin PSP-I were used. The models revealed the catalytic and substrate-binding sites and the negatively charged residue responsible for the trypsin-like activity. IgE-binding and antigenic sites in the two allergens were predicted using the models and programs based on the structure of known epitopes. Api SI and Api SII show structural and functional similarity to the members of the PPAF-II family. Most probably, they are part of the defence system of Apis mellifera

Bioinformatic searches of genome databases revealed that the number of autophagy-related genes (ATG) is considerably lower in trypanosomes than in higher eukaryotes and even in yeast. This raises the question of whether autophagy in this protozoan parasite is more primitive and represents a rudimentary paradigm due to its early branching off the evolutionary tree. We here present the crystal structure of TbATG8B. This molecule (MW 13.7 kDa) belongs to the ubiquitin-like proteins showing the typical ubiquitin fold and strong sequence homology to LC3, the human homologue. Due to its characteristic folding, it should readily bind to TbATG4.1 for being processed. This presumption was tested by molecular modeling approaches, docking TbATG8B to a homology model of TbATG4.1. Although exchanges of several amino acids are evident from sequence comparisons, the overall structure seems very much alike and the necessary catalytic triad (C-D-H) is well conserved in TbATG4.1. Thus membrane formation during appearance of the autophagic bodies seems very similar in trypanosomes and their higher eukaryotic counterpart.

The homochirality of biomolecules is a prerequisite for the origin and evolution of terrestrial life. The unique selection of D-monosaccharides, in particular, D-ribose in RNA and D-deoxyribose in DNA, leads to the construction of proteins by L-amino acids. This points to the exclusive role of stereoselectivity in the most important physiological processes. So far, there is no experimental confirmation for the theoretical calculations of the energy differences between enantiomers used for the explanation of the stereoselection of biomolecules. Therefore, the question of why nature prefers one configuration over the other still lacks a definitive answer. Here, we present the first experimental evidence that the D-enantiomer of RNA has a different electronic structure compared to the corresponding L-enantiomer. When varying the incident photon energy of the ultraviolet Raman probe across 5 eV, D- and L-isomers of the RNA duplex with the sequence [r(CUGGGCGG).r(CCGCCUGG)] show differences in the intensity of the vibrational modes with energies of 124.0 meV to 210.8 meV. The intensity difference of these vibrational modes can be traced back to energy differences in the electronic levels of D- and L-RNA leading to the preferential stabilization of the naturally occurring D-configuration of RNA over the L-configuration.

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasianahemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasianahemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.

The neurotoxin vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis. It is a heterodimer of a basic toxic His-48 phospholipase A2 (PLA2) and an acidic nontoxic Gln-48 PLA2. The shape of the neurotoxin and its separated components in solution as well as their interactions with calcium, the brain phospholipid phosphatidylcholine, and two inhibitors, elaidoylamide and vitamin E, were investigated by dynamic light scattering. Calcium binding is connected with a conformational change in vipoxin observed as a change of the hydrodynamic shape from oblate ellipsoid to a shape closer to a sphere. The Ca2+-bound form of vipoxin, which is catalytically active, is more compact and symmetric than the calcium-free heterodimer. Similar changes were observed as a result of the Ca2+-binding to the two separated subunits. In the presence of aggregated phosphatidylcholine, the neurotoxic complex dissociates to subunits. It is supposed that only the toxic component binds to the substrate, and the other subunit, which plays a chaperone function, remains in solution. The inhibition of vipoxin with the synthetic inhibitor elaidoylamide and the natural compound vitamin E changes the shape of the toxin from oblate to prolate ellipsoid. The inhibited toxin is more asymmetric in comparison to the native one. Similar, but not so pronounced, effects were observed after the inhibition of the monomeric and homodimeric forms of the toxic His-48 PLA2. Circular dichroism measurements in the presence of urea, methylurea, and ethylurea indicate a strong hydrophobic stabilization of the neurotoxin. Hydrophobic interactions stabilize not only the folded regions but also the regions of intersubunit contacts.

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNAMetbut not for yeast tRNAMetior E. colitRNAMetf. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNAMetisusceptible to the specific cleavage reaction. Using crystallographic data of tRNAMetfof E. coliwith U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNAMetimade changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.

Vipoxin from the venom of Vipera ammodytes meridionalis is an unique neurotoxic complex between a toxic phospholipase A2 and a highly homologous non-toxic protein inhibitor. It is an example of evolution of a catalytic and toxic function into inhibitory and non-toxic one. The activity of the V. ammodytes meridionalis toxin is 1.7 times higher than that of the closely related (92% sequence identity) neurotoxic complex RV4/RV7 from the venom of Vipera russelli formosensis. The enhanced enzymatic activity of vipoxin is attributed to limited structural changes, in particular to the substitutions G54R and Q78K in the PLA2 subunit of the complex and to the T54R substitution in the inhibitor.

From mistletoe Viscum album L. extracts three chitin-binding lectin isoforms, cbML1, cbML2, and cbML3, were isolated and their primary structure determined. All three cbML isoforms are composed of two protein chains of 48 or 49 amino acid residues, linked by an intermolecular disulfide bond. The sequence of each single cbML chain is characterized by a relatively high number of cysteine and glycine residues, 9 and 6, respectively, and contains four intramolecular disulfide bridges. On the basis of the combined interpretation of sequencing and MALDI MS data, the following results for the three cbML isoforms were obtained: the first one consists of two identical truncated polypeptide chains (1–48), the second is a heterodimer, containing one truncated (1–48) and one full-length chain (1–49), and the third is composed of two full length chains (1–49). The cbML sequence shows 55% identity to hevein, a single-chain chitin-binding protein of 43 amino acids, one of the most predominant proteins in natural rubber latex. On the basis of the NMR data on hevein from Hevea brasiliensis the three-dimensional structure of cbML3 was modelled. The 26 sequence changes between cbML3 and hevein were accommodated with only little perturbation in the main chain folding. A comparison of the primary structures of cbML3 and hevein is shown and the effects of the sequence changes are discussed. Differences have been identified in the loop region of the molecule and the potential interface region of cbML3 supporting the dimer formation. The high-affinity chitin-binding site seems to be highly conserved.

The structure of native yeast tRNAPheand wheat germ ribosomal 5S RNA induced by different magnesium ion concentrations was studied in solution with a synchrotron mediated hydroxyl radical RNA cleavage reaction. We showed that very small amounts of Mg+2can induce significant changes in the hydroxyl radical cleavage pattern of tRNAPhe. It also turned out that a reactivity of tRNAPhetowards •OH coincides with the strong metal binding sites. Because of the Mg ions are heavily hydrated one can suggest the strong correlation of the observed nucleosides reactivity in vicinity of Mg2+binding sites with availability of water molecules as a source of hydroxyl radical. On the other hand the structure of wheat germ 5S rRNA is less sensitive to the hydroxyl radical reaction than tRNAPhealthough some changes are visible at 4 mM Mg ions. It is probably due to the lack of strong Mg+2binding sites in that molecule. The reactivity of nucleotides in loops C and D of 5S rRNA is not effected, what suggests their flexibility or involvement in higher order structure formation. There is different effect of magnesium on tRNA and 5S rRNA folding. We found that nucleotides forming strong binding sites for magnesium are very sensitive to X-ray generated hydroxyl radical and can be mapped with •OH. The results show, that guanine nucleotides are preferentially hydrated. X-ray footprinting mediated hydroxyl radical RNA cleavage is a very powerful method and has been applied to studies of stable RNAs for the first time.

Savinase and Esperase are closely related highly alkalophilic proteinases produced by Bacillus lentus. They are suitable couple for investigating the structural basis of proteinase specificity due to the identity of the catalytic and the differences in the substrate binding sites. Two of the substitutions in these sites are very important: T129P and G131P. The two prolines provide an extra rigidity of the Savinase-binding site. The substitutions S166N and Q191T in the S1 recognition loop change the binding geometry of the substrate P1 residue. The geometry of S1 in Esperase is more favorable for binding and catalysis in comparison to that in Savinase. Differences in P3 specificity are probably created by the substitution V104L, which influences the conformation of S3. Leu in position 104 is more favorable for the binding of Phe to S4 than Val. The lower affinity and catalytic efficiency as well as more narrow proteolytic specificity of Savinase in comparison to those of Esperase are explained with the extra rigidity and unfavorable changes in geometry of the substrate binding site of the first enzyme.

The shape of free Thermus flavus5 S rRNA in solution at 1.3 nm resolution is restored from synchrotron x-ray scattering data using an ab initiosimulated annealing algorithm. The free 5 S rRNA is a bent elongated molecule displaying a compact central region and two projecting arms, similar to those of the tRNA. The atomic models of the 5 S rRNA domains A-D-E and B-C in the form of elongated helices can be well accommodated within the shape, yielding a tentative model of the structure of the free 5 S rRNA in solution. Its comparison with the recent protein-RNA map in the ribosome (Svergun, D. I., and Nierhaus, K. H. (2000) J. Biol. Chem.275, 14432–14439) indicates that the 5 S rRNA becomes essentially more compact upon complex formation with specific ribosomal proteins. A conceivable conformational change involves rotation of the B-C domain toward the A-D-E domain. The model of free 5 S rRNA displays no interactions between domains E and C, but such interactions are possible in the bound molecule.

The crystal structure of the ribosome-inactivating protein (RIP) mistletoe lectin I (ML-I) fromViscum albumhas been solved by molecular replacement techniques. The structure has been refined to a crystallographic R-factor of 24.5% using X-ray diffraction data to 2.8 Å resolution. The heterodimeric 63-kDa protein consists of a toxic A subunit which exhibits RNA-glycosidase activity and a galactose-specific lectin B subunit. The overall protein fold is similar to that of ricin fromRicinus communis;however, unlike ricin, ML-I is already medically applied as a component of a commercially available misteltoe extract with immunostimulating potency and for the treatment of human cancer. The three-dimensional structure reported here revealed structural details of this pharmaceutically important protein. The comparison to the structure of ricin gives more insights into the functional mechanism of this protein, provides structural details for further protein engineering studies, and may lead to the development of more effective therapeutic RIPs.

The structure of the peroxisomal catalase A from the budding yeast Saccharomyces cerevisiae, with 515 residues per subunit, has been determined and refined to 2.4 A˚ resolution. The crystallographic agreement factors R and Rfree are 15.4 % and 19.8 %, respectively. A tetramer with accurate 222-molecular symmetry is located in the asymmetric unit of the crystal. The conformation of the central core of catalase A, about 300 residues, remains similar to the structure of catalases from distantly related organisms. In contrast, catalase A lacks a carboxy-terminal domain equivalent to that found in catalase from Penicillium vitalae, the only other fungal catalase structure available. Structural peculiarities related with the heme and NADP(H) binding pockets can be correlated with biochemical characteristics of the catalase A enzyme. The network of molecular cavities and channels, filled with solvent molecules, supports the existence of one major substrate entry and at least two possible alternative pathways to the heme active site. The structure of the variant protein Val111Ala, also determined by X-ray crystallography at 2.8 A˚ resolution, shows a few, well-localized, differences with respect to the wild-type enzyme. These differences, that include the widening of the entry channel in its narrowest point, provide an explanation for both the increased peroxidatic activity and the reduced catalatic activity of this mutant.

Bacitracins are a group of widely used peptide antibiotics. There has been interest in determining the three-dimensional structure of the bacitracins. However, solution studies indicate significant flexibility in their structure and to date native bacitracins have resisted attempts at crystallisation despite considerable efforts over a number of years by several groups. Here we report the first three-dimensional X-ray structure of a bacitracin, complexed to a subtilisin proteinase. X-Ray diffraction data were collected using synchrotron radiation in combination with the Image Plate Scanner system. The complex structure including two enzymes, two bacitracins, 220 water molecules and two Ca 2+ions was refined by restrained least-squares to a crystallographic Rfactor (=Σ{{ Fo- Fc}}/Σ{ Fo}}) of 16.3% at 2.0 Å.

The major protein component in seeds is storage protein. These have no known enzymatic activity and act to provide amino acids as a source of metabolites in the developing seedling. We report here the first three dimensional crystal structure of a seed storage globulin at high resolution. The molecule of the 2S globulin, narbonin, from Vicia narbonensisL. consists of an eight-stranded parallel α/gb barrel structure similar to that observed in triose phosphate isomerase (TIM). Narbonin is the first protein with this topology possessing no known enzymatic activity. Because of the lack of sequence information most of the primary structure was determined directly from the electron density.

Proteinase K (EC 3.4.21.14) from the fungus Tritirachium albumLimber is the most active known serine endopeptidase. The sequence of its 275-residue long polypeptide chain and its three-dimensional folding show a high degree of homology with the bacterial subtilisin proteases. Using difference Fourier methods, the binding mode of the synthetic carbobenzoxy-Ala-Ala-chloromethyl ketone inhibitor to the active site of proteinase K was determined. In several cycles of restrained least-squares, the enzyme-inhibitor complex was refined to a current R= 12% for 9400 X-ray diffraction data between 2.2 and 5.0 Å resolution. The inhibitor is attached to proteinase K by two covalent bonds; one between the methylene carbon of the inhibitor and Nϵ2 of the catalytic His 68, the other between the ketone carbon atom of the inhibitor and Oγ of the catalytic Ser 221. In addition, two hydrogen bonds donated by the peptide NH of Ser 221 and by the side chain NH 2of Asn 160 hold the hemiketal O −in the oxyanion hole. The peptide inhibitor is further hydrogen bonded to the proteinase polypeptide chain in a three-stranded antiparallel pleated sheet.

One of the three wheat germ inhibitors of proteinase K is bifunctional and inhibits simultaneously proteinase K (or subtilisin but not enzymes of the trypsin family) and insect α-amylase. The molecular mass of this inhibitor called PKI-3 is 21 kDa, and the binding constant for proteinase K is 0.8 nM at pH 8.2, 25°C, in 1:1 molar ratio. PKI-3 was crystallized by microdialysis against 10–12% polyethylene glycol 6000, 50 mM NaH 2PO 4, pH 6.7. The crystals have monoclinic space group P2 1with a= 42.5, b= 65.3, c= 31.5 Å, β = 110°, and diffract beyond 2.0 Å resolution. The complex proteinase K · PKI-3 was crystallized by equilibrium vapor diffusion under the same conditions. The crystals are needle-shaped and still too small for X-ray analysis. Gel electrophoresis established the composition of the crystals.

a-Cyclodextrin, a torus shaped molecule with a 5 Å wide central cavity, forms a number of deep green, blue and black crystals when complexed with iodine/metal iodide. In contrast, ß-cyclodextrin, having a 6 Å cavity produces only one type of reddish-brown crystal, no matter what metal iodide is used. The complex (ß-cyclodextrin)2 ·KI7·9H2O displays space groupP21 (pseudo-C2) with cell constantsa=19.609(5),b=24.513(7),c=15.795(6)Å, ß=109.50(2)°,Z=4. The crystal structure was solved inC2 on the basis of 3022 absorption corrected CuKa (Ni-filter) X-ray intensities and refined by full matrix least squares toR=17%. This relatively highR-factor is due to many weak reflections (pseudo-C2) and considerable disorder exhibited by water and iodine. In the complex, ß-cyclodextrin adopts a ‘round’ shape with O(2)...O(3) interglucose hydrogen bonds formed and all O(6) hydroxyls pointing away from the cavity. Two molecules are arranged head-to-head to produce a dimer, and dimers are stacked such that a slightly zigzagged cylinder with a 6 Å-wide cavity is formed. In the cavity described by each dimer, an I7- ion composed of I2·I3-·I2 units is located, with I2 and I3- perpendicular to each other. K+ ions and 9 H2O molecules are found in interstices between the ß-cyclodextrin cylinders. This zigzag polyiodide contrasts with the linear form observed in the 5 Å wide a-cyclodextrin channels. It explains differences in color of the crystals and suggests that ß-cyclodextrin polyiodide is not a good model for blue starch-iodine.

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N‐terminal and C‐terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C‐ and N‐lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N‐lobe and C‐lobe showed that the inhibitory fragment came from the C‐lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H2N‐Val‐Ala‐Gln‐Gly‐Ala‐Ala‐Gly‐Leu‐Ala‐COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P21with cell dimensions a = 44.4 Å, b = 38.6 Å, c = 79.2 Å, β = 105.8oand Z = 2. The crystal structure has been determined at 2.4 Å resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf‐fragment forms several intermolecular interactions with proteinase K. The Ser‐224 Oγ and His‐57 Nϵ2 move away to a distance of 3.68 Å in the complex. In the crystal structure, Gln‐3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp‐254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat. Proteins 33:30–38, 1998. © 1998 Wiley‐Liss, Inc.

The first three-dimensional structure of the ribosome inactivating protein mistletoe lectin I (ML-I) fromViscum albumhas been modeled on the basis of the X-ray structure of castor bean ricin fromRicinus communis.The relative high sequence homology and conserved secondary structure enabled accurate modeling. The 196 sequence changes between ML-I and ricin could be accomodated with only little pertubation in the main chain folding. A close comparison of the primary structures of ML-I and ricin is given and the effects of the sequence changes are elucidated on the basis of the modeled three-dimensional structure. Differences have been identified in the vicinity of the active site, in the high affinity galactose binding site and in the interface between the A and B chains, which might account for the reduced cytotoxicity of ML-I.

Group V major allergen Phl p 5b of timothy grass pollen induces allergic rhinitis and bronchial asthma in 90% of grass pollen-allergic patients. In addition to its allergenicity ribonuclease activity has recently been attributed to this 29-kDa protein.

Thermitase, the thermostable alkaline protease from Thermoactinomyces vulgaris, has been crystallised in a 1:1 complex with eglin, the inhibitor from the medical leech. Two large crystals were grown, with cell dimensions of a= 49.3 Å, b= 67.3 Å, c= 90.5 Å and space group P2 12 12 1. The crystals are relatively tightly packed with Vm= 2.1 Å 3/Da. Three-dimensional data to 1.9 Å have been recorded from one of these crystals. The orientation and position of the complex in the unit cell have been established using the subtilisin Carlsberg-eglin structure as a model. The structure of the complex is being refined by restrained least-squares. The present crystallographic Rfactor (= Σ| Fo− Fc|/Σ| Fo) is 26% at 2.5 Å resolution.

Many attempts have been made to elucidate the three-dimensional structure from elongation factor Tu, but so far the only crystals suitable for X-ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF-Tu from Thermus aquaticus. The crystals belong to hexagonal space group P6 322 and diffract up to 2.6 Å. The cell dimensions are a= b= 178 Å, c= 238 Å and 6 molecules are contained per asymmetric unit.

For the crystallization of proteins under microgravity conditions, a Chinese re-entry system was used, in which 101 experiments of 25 different biological macromolecules were accommodated. From the results obtained we conclude that under microgravity conditions crystal growth can only be expected under those crystallization conditions which also permit crystal growthon earth. A number of space-grown crystals were larger in size and of a better quality in their ability to diffract X-rays than the corresponding ground control crystals grown at the Chinese launch site. However, the space-grown crystals have not reached the X-ray diffraction quality of the crystals obtained under optimal conditions in the home laboratories.

The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three-dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the ‘scissile’ bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%.

Thermitase, the thermostable alkaline protease from Thermoactinomyces vulgaris, has been crystallised in a 1:1 complex with eglin, the inhibitor from the medical leech. Two large crystals were grown, with cell dimensions of a= 49.3 Å, b= 67.3 Å, c= 90.5 Å and space group P212121. The crystals are relatively tightly packed with Vm= 2.1 Å3/Da. Three‐dimensional data to 1.9 Å have been recorded from one of these crystals. The orientation and position of the complex in the unit cell have been established using the subtilisin Carlsberg‐eglin structure as a model. The structure of the complex is being refined by restrained least‐squares. The present crystallographic Rfactor (= Σ|Fo−Fc|/Σ|Fo) is 26% at 2.5 Å resolution.

Many attempts have been made to elucidate the three‐dimensional structure from elongation factor Tu, but so far the only crystals suitable for X‐ray crystallography contained a partially degraded protein. Here, we report the crystallization of a fully active, intact EF‐Tu from Thermus aquaticus. The crystals belong to hexagonal space group P6322 and diffract up to 2.6 Å. The cell dimensions are a= b= 178 Å, c= 238 Å and 6 molecules are contained per asymmetric unit.

Lactoferrin is an iron binding glycoprotein with a molecular weight of 80 kDa. The molecule is divided into two lobes representing the N-terminal and C-terminal halves of the polypeptide chain, each containing an iron binding site. The serine proteinases such as trypsin, chymotrypsin, and pepsin hydrolyze lactoferrin into two unequal halves while proteinase K divides this protein into two equal halves. In the first step of hydrolysis by proteinase K, the C- and N-lobes, each having a molecular weight of approximately 40 kDa, are generated. In the next step, the lobes are further hydrolyzed into small molecular weight peptides. The proteinase K isolated from the hydrolyzed product does not show enzymatic activity suggesting that the enzyme is inhibited. Furthermore, the hydrolysis experiments on N-lobe and C-lobe showed that the inhibitory fragment came from the C-lobe. The purified lactoferrin fragment was found to be a decapeptide with an amino acid sequence of H2N-Val-Ala-Gln-Gly-Ala-Ala-Gly-Leu-Ala-COOH. The complex formed between proteinase K and lactoferrin fragment was crystallized by microdialysis. The crystals belonged to the monoclinic space group P21with cell dimensions a = 44.4 Å, b = 38.6 Å, c = 79.2 Å, β = 105.8^o and Z = 2. The crystal structure has been determined at 2.4 Å resolution. It has been refined to an R factor of 0.163 for 9044 reflections. The Lf-fragment forms several intermolecular interactions with proteinase K. The Ser-224 Oγand His-57 Nε2 move away to a distance of 3.68 Å in the complex. In the crystal structure, Gln-3I (I indicates inhibitor i.e., lactoferrin fragment) is involved in a direct intermolecular interaction with a symmetry related proteinase K molecule through a strong hydrogen bond with Asp-254. The mode of intermolecular interactions in the complex conformational features of the enzyme and placement of the fragment with respect to the enzyme resemble with the molecular complex of proteinase K with its natural inhibitor PKI3 from wheat. Proteins 33:30–38, 1998. © 1998 Wiley-Liss, Inc.

Proteinase K (EC 3.4.21.14) from the fungus Tritirachium albumLimber is the most active known serine endopeptidase. The sequence of its 275‐residue long polypeptide chain and its three‐dimensional folding show a high degree of homology with the bacterial subtilisin proteases. Using difference Fourier methods, the binding mode of the synthetic carbobenzoxy‐Ala‐Ala‐chloromethyl ketone inhibitor to the active site of proteinase K was determined. In several cycles of restrained least‐squares, the enzyme‐inhibitor complex was refined to a current R= 12% for 9400 X‐ray diffraction data between 2.2 and 5.0 Å resolution. The inhibitor is attached to proteinase K by two covalent bonds; one between the methylene carbon of the inhibitor and Nϵ2 of the catalytic His 68, the other between the ketone carbon atom of the inhibitor and Oγ of the catalytic Ser 221. In addition, two hydrogen bonds donated by the peptide NH of Ser 221 and by the side chain NH2of Asn 160 hold the hemiketal O−in the oxyanion hole. The peptide inhibitor is further hydrogen bonded to the proteinase polypeptide chain in a three‐stranded antiparallel pleated sheet.

One of the three wheat germ inhibitors of proteinase K is bifunctional and inhibits simultaneously proteinase K (or subtilisin but not enzymes of the trypsin family) and insect α‐amylase. The molecular mass of this inhibitor called PKI‐3 is 21 kDa, and the binding constant for proteinase K is 0.8 nM at pH 8.2, 25°C, in 1:1 molar ratio. PKI‐3 was crystallized by microdialysis against 10–12% polyethylene glycol 6000, 50 mM NaH2PO4, pH 6.7. The crystals have monoclinic space group P21with a= 42.5, b= 65.3, c= 31.5 Å, β = 110°, and diffract beyond 2.0 Å resolution. The complex proteinase K · PKI‐3 was crystallized by equilibrium vapor diffusion under the same conditions. The crystals are needle‐shaped and still too small for X‐ray analysis. Gel electrophoresis established the composition of the crystals.

Bacitracins are a group of widely used peptide antibiotics. There has been interest in determining the three‐dimensional structure of the bacitracins. However, solution studies indicate significant flexibility in their structure and to date native bacitracins have resisted attempts at crystallisation despite considerable efforts over a number of years by several groups. Here we report the first three‐dimensional X‐ray structure of a bacitracin, complexed to a subtilisin proteinase. X‐Ray diffraction data were collected using synchrotron radiation in combination with the Image Plate Scanner system. The complex structure including two enzymes, two bacitracins, 220 water molecules and two Ca2+ions was refined by restrained least‐squares to a crystallographic Rfactor (=Σ{{Fo‐Fc}}/Σ{Fo}}) of 16.3% at 2.0 Å.

The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure. We have introduced specific mutations (T71l, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase™ from Bacillus lentus. This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel. The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule. The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.

The major protein component in seeds is storage protein. These have no known enzymatic activity and act to provide amino acids as a source of metabolites in the developing seedling. We report here the first three dimensional crystal structure of a seed storage globulin at high resolution. The molecule of the 2S globulin, narbonin, from Vicia narbonensisL. consists of an eight‐stranded parallel α/gb barrel structure similar to that observed in triose phosphate isomerase (TIM). Narbonin is the first protein with this topology possessing no known enzymatic activity. Because of the lack of sequence information most of the primary structure was determined directly from the electron density.

For the crystallization of proteins under microgravity conditions, a Chinese re‐entry system was used, in which 101 experiments of 25 different biological macromolecules were accommodated. From the results obtained we conclude that under microgravity conditions crystal growth can only be expected under those crystallization conditions which also permit crystal growthon earth. A number of space‐grown crystals were larger in size and of a better quality in their ability to diffract X‐rays than the corresponding ground control crystals grown at the Chinese launch site. However, the space‐grown crystals have not reached the X‐ray diffraction quality of the crystals obtained under optimal conditions in the home laboratories.