In vivo imaging of CNS microglial activation/macrophage infiltration with combined [18F]DPA-714-PET and SPIO-MRI in a mouse model of relapsing remitting experimental autoimmune encephalomyelitis.

PURPOSE: To evaluate the feasibility and sensitivity of multimodality PET/CT and MRI imaging for non-invasive characterization of brain microglial/macrophage activation occurring during the acute phase in a mouse model of relapsing remitting multiple sclerosis (RR-MS) using [18F]DPA-714, a selective radioligand for the 18-kDa translocator protein (TSPO), superparamagnetic iron oxide particles (SPIO), and ex vivo immunohistochemistry. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in female SJL/J mice by immunization with PLP139-151. Seven symptomatic EAE mice and five controls underwent both PET/CT and MRI studies between 11 and 14 days post-immunization. SPIO was injected i.v. in the same animals immediately after [18F]DPA-714 and MRI acquisition was performed after 24 h. Regional brain volumes were defined according to a mouse brain atlas on co-registered PET and SPIO-MRI images. [18F]DPA-714 standardized uptake value (SUV) ratios (SUVR), with unaffected neocortex as reference, and SPIO fractional volumes (SPIO-Vol) were generated. Both SUVR and SPIO-Vol values were correlated with the clinical score (CS) and among them. Five EAE and four control mice underwent immunohistochemical analysis with the aim of identifying activated microglia/macrophage and TSPO expressions. RESULTS: SUVR and SPIO-Vol values were significantly increased in EAE compared with controls in the hippocampus (p < 0.01; p < 0.02, respectively), thalamus (p < 0.02; p < 0.05, respectively), and cerebellum and brainstem (p < 0.02), while only SPIO-Vol was significantly increased in the caudate/putamen (p < 0.05). Both SUVR and SPIO-Vol values were positively significantly correlated with CS and among them in the same regions. TSPO/Iba1 and F4/80/Prussian blue staining immunohistochemistry suggests that increased activated microglia/macrophages underlay TSPO expression and SPIO uptake in symptomatic EAE mice. CONCLUSIONS: These preliminary results suggest that both activated microglia and infiltrated macrophages are present in vulnerable brain regions during the acute phase of PLP-EAE and contribute to disease severity. Both [18F]DPA-714-PET and SPIO-MRI appear suitable modalities for preclinical study of neuroinflammation in MS mice models.

Imaging of brain TSPO expression in a mouse model of amyotrophic lateral sclerosis with (18)F-DPA-714 and micro-PET/CT.

PURPOSE: To evaluate the feasibility and sensitivity of (18)F-DPA-714 for the study of microglial activation in the brain and spinal cord of transgenic SOD1(G93A) mice using high-resolution PET/CT and to evaluate the Iba1 and TSPO expression with immunohistochemistry. METHODS: Nine symptomatic SOD1(G93A) mice (aged 117 ± 12.7 days, clinical score range 1 - 4) and five WT SOD1 control mice (aged 108 ± 28.5 days) underwent (18)F-DPA-714 PET/CT. SUV ratios were calculated by normalizing the cerebellar (rCRB), brainstem (rBS), motor cortex (rMCX) and cervical spinal cord (rCSC) activities to that of the frontal association cortex. Two WT SOD1 and six symptomatic SOD1(G93A) mice were studied by immunohistochemistry. RESULTS: In the symptomatic SOD1(G93A) mice, rCRB, rBS and rCSC were increased as compared to the values in WT SOD1 mice, with a statistically significantly difference in rBS (2.340 ± 0.784 vs 1.576 ± 0.287, p = 0.014). Immunofluorescence studies showed that TSPO expression was increased in the trigeminal, facial, ambiguus and hypoglossal nuclei, as well as in the spinal cord, of symptomatic SOD1(G93A) mice and was colocalized with increased Iba1 staining. CONCLUSION: Increased (18)F-DPA-714 uptake can be detected with high-resolution PET/CT in the brainstem of transgenic SOD1(G93A) mice, a region known to be a site of degeneration and increased microglial activation in amyotrophic lateral sclerosis, in agreement with increased TSPO expression in the brainstem nuclei shown by immunostaining. Therefore, (18)F-DPA-714 PET/CT might be a suitable tool to evaluate microglial activation in the SOD1(G93A) mouse model.

A randomized controlled double-blind investigation of the effects of vitamin D dietary supplementation in subjects with atopic dermatitis.

BACKGROUND: Subjects with atopic dermatitis (AD) have defects in antimicrobial peptide (AMP) production possibly contributing to an increased risk of infections. In laboratory models, vitamin D can alter innate immunity by increasing AMP production. OBJECTIVE: To determine if AD severity correlates with baseline vitamin D levels, and to test whether supplementation with oral vitamin D alters AMP production in AD skin. METHODS: This was a multi-centre, placebo-controlled, double-blind study in 30 subjects with AD, 30 non-atopic subjects, and 16 subjects with psoriasis. Subjects were randomized to receive either 4000 IU of cholecalciferol or placebo for 21 days. At baseline and day 21, levels of 25-hydroxyvitamin D (25OHD), cathelicidin, HBD-3, IL-13, and Eczema Area and Severity Index (EASI) and Rajka-Langeland scores were obtained. RESULTS: At baseline, 20% of AD subjects had serum 25OHD below 20 ng/mL. Low serum 25OHD correlated with increased Fitzpatrick Skin Type and elevated BMI, but not AD severity. After 21 days of oral cholecalciferol, mean serum 25OHD increased, but there was no significant change in skin cathelicidin, HBD-3, IL-13 or EASI scores. CONCLUSIONS: This study illustrated that darker skin types and elevated BMI are important risk factors for vitamin D deficiency in subjects with AD, and highlighted the possibility that seasonality and locale may be potent contributors to cathelicidin induction through their effect on steady state 25OHD levels. Given the molecular links between vitamin D and immune function, further study of vitamin D supplementation in subjects with AD is warranted.

Peripheral blood gene expression in alopecia areata reveals molecular pathways distinguishing heritability, disease and severity.

Alopecia areata (AA) is an autoimmune hair loss disorder in which systemic disturbances have been described, but are poorly understood. To evaluate disease mechanisms, we examined gene expression in the blood of defined clinical subgroups (patchy AA persistent type, AAP, n=5; alopecia universalis, AU, n=4) and healthy controls (unaffected relatives, UaR, n=5; unaffected non-relatives, UaNR, n=4) using microarrays. Unsupervised hierarchical clustering separates all four patient and control groups, producing three distinct expression patterns reflective of 'inheritance', 'disease' and 'severity' signatures. Functional classification of differentially expressed genes (DEGs) comparing disease (AAP, AU) vs normal (UaR) groups reveals upregulation in immune response, cytokine signaling, signal transduction, cell cycle, proteolysis and cell adhesion-related genes. Pathway analysis further reveals the activation of several genes related to natural killer-cell cytotoxicity, apoptosis, mitogen activated protein kinase, Wnt signaling and B- and T-cell receptor signaling in AA patients. Finally, 35 genes differentially expressed in AA blood overlap with DEGs previously identified in AA skin lesions. Our results implicate innate and adaptive immune processes while also revealing novel pathways, such as Wnt signaling and apoptosis, relevant to AA pathogenesis. Our data suggest that peripheral blood expression profiles of AA patients likely carry new biomarkers associated with disease susceptibility and expression.

Structure of a complex of two plasma proteins: transthyretin and retinol-binding protein.

The three-dimensional structure of the complex formed by two plasma proteins, transthyretin and retinol-binding protein, was determined from x-ray diffraction data to a nominal resolution of 3.1 angstroms. One tetramer of transthyretin was bound to two molecules of retinol-binding protein. The two retinol-binding protein molecules established molecular interactions with the same transthyretin dimer, and each also made contacts with one of the other two monomers. Thus, the other two potential binding sites in a transthyretin tetramer were blocked. The amino acid residues of the retinol-binding protein that were involved in the contacts were close to the retinol-binding site.

A novel deamido-NAD+-binding site revealed by the trapped NAD-adenylate intermediate in the NAD+ synthetase structure.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) has a central role in life processes. The ubiquitous enzyme NAD+ synthetase catalyzes a key step in NAD+ biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction. NAD+ synthetase belongs to the amidotransferase family and has been recognized as a member of the family of N-type ATP pyrophosphatases. In order to investigate the mechanism of the reaction carried out by NAD+ synthetase we have determined a high-resolution three-dimensional structure of the Bacillus subtilis homodimeric NAD+ synthetase in complex with the trapped reaction intermediate NAD-adenylate. RESULTS: Two NAD-adenylate molecules and two pyrophosphate (PPi) molecules are observed in the 1.3 A resolution structure of the NAD+ synthetase-NAD-adenylate complex. Structural studies on the NAD+ synthetase-NAD-adenylate adduct and on the cation-binding sites reveal a new deamido-NAD+-binding site located at the subunit interface, locate a binuclear magnesium cluster at the ATP-binding site and, identify two monovalent cation sites, one of which may represent an ammonium-binding site. CONCLUSIONS: Our results suggest that two different catalytic strategies have been adopted by NAD+ synthetase in the two different steps of the reaction. During the adenylation step, no protein residues seem to be located properly to directly participate in catalysis, which is likely to be carried out with the fundamental assistance of an electron-withdrawing trimetallic constellation present in the active site. A different behavior is observed for the second step, in which an ammonium ion is the binding species. In this step, Asp173 is a key residue in both deprotonation of the primarily bound ammonium ion, and stabilization of the tetrahedral transition-state intermediate. Moreover, the structural data suggest that product release can take place only after all substrates are bound to the enzyme, and product release is ultimately controlled by the conformation adopted by two mobile loops.

Unusual structure of the oxygen-binding site in the dimeric bacterial hemoglobin from Vitreoscilla sp.

BACKGROUND: The first hemoglobin identified in bacteria was isolated from Vitreoscilla stercoraria (VtHb) as a homodimeric species. The wild-type protein has been reported to display medium oxygen affinity and cooperative ligand-binding properties. Moreover, VtHb can support aerobic growth in Escherichia coli with impaired terminal oxidase function. This ability of VtHb to improve the growth properties of E. coli has important applications in fermentation technology, assisting the overexpression of recombinant proteins and antibiotics. Oxygen binding heme domains have been identified in chimeric proteins from bacteria and yeast, where they are covalently linked to FAD- and NAD(P)H-binding domains. We investigate here the fold, the distal heme site structure and the quaternary assembly of a bacterial hemoglobin which does not bear the typical flavohemoglobin domain organization. RESULTS: The VtHb three-dimensional structure conforms to the well known globin fold. Nevertheless, the polypeptide segment connecting helices C and E is disordered, and residues E7-E10 (defined according to the standard globin fold nomenclature) do not adopt the usual alpha-helical conformation, thus locating Gln53(E7) out of the heme pocket. Binding of azide to the heme iron introduces substantial structural perturbations in the heme distal site residues, particularly Tyr29(B10) and Pro54(E8). The quaternary assembly of homodimeric VtHb, not observed before within the globin family, is based on a molecular interface defined by helices F and H of both subunits, the two heme iron atoms being 34 A apart. CONCLUSIONS: The unusual heme distal site structure observed shows that previously undescribed molecular mechanisms of ligand stabilization are operative in VtHb. The polypeptide chain disorder observed in the CE region indicates a potential site of interaction with the FAD/NADH reductase partner, in analogy with observations in the chimeric flavohemoglobin from Alcaligenes eutrophus.

A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase.

BACKGROUND: Polyamines are essential for cell growth and differentiation; compounds interfering with their metabolism are potential anticancer agents. Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary amino groups of spermine and spermidine. RESULTS: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Angstroms. PAO from Zea mays contains two domains, which define a remarkable 30 Angstrom long U-shaped catalytic tunnel at their interface. The structure of PAO in complex with the inhibitor MDL72527 reveals the residues forming the catalytic machinery and unusual enzyme-inhibitor CH.O H bonds. A ring of glutamate and aspartate residues surrounding one of the two tunnel openings contributes to the steering of the substrate towards the inside of the tunnel. CONCLUSIONS: PAO specifically oxidizes substrates that have both primary and secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with respect to the flavin. Conservation of an N-terminal sequence motif indicates that PAO is member of a novel family of flavoenzymes. Among these, monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.

Structure of L-aspartate oxidase: implications for the succinate dehydrogenase/fumarate reductase oxidoreductase family.

BACKGROUND: Given the vital role of NAD+ in cell metabolism, the enzymes involved in bacterial de novo NAD+ biosynthesis are possible targets for drug design against pathogenic bacteria. The first reaction in the pathway is catalysed by L-aspartate oxidase (LASPO), a flavoenzyme that converts aspartate to iminoaspartate using either molecular oxygen or fumarate as electron acceptors. LASPO has considerable sequence homology with the flavoprotein subunits of succinate dehydrogenase (SDH) and fumarate reductase (FRD). RESULTS: The crystal structure of the apoform of LASPO from Escherichia coli has been determined to 2.2 A resolution. The enzyme shows a novel fold for an FAD-dependent protein, comprising a three-domain structure: an FAD-binding domain with the dinucleotide-binding fold, a C-terminal three-helical bundle domain, and an alpha + beta capping domain, which is topologically similar to the small subunit of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase. The interface between the FAD-binding and capping domains defines a cleft in which the active site is located. CONCLUSIONS: A number of strictly conserved residues present in all three domains indicate that LASPO, SDH and FRD share the same overall folding topology. Many of these conserved residues are in the FAD-binding site and active centre, suggesting a similar catalytic mechanism. Thus, LASPO, SDH and FRD form a class of functionally and structurally related oxidoreductases that are all able to reduce fumarate and to oxidise a dicarboxylate substrate.

Crystal structure of Escherichia coli pyruvate kinase type I: molecular basis of the allosteric transition.

BACKGROUND: Pyruvate kinase (PK) plays a major role in the regulation of glycolysis. Its catalytic activity is controlled by the substrate phosphoenolpyruvate and by one or more allosteric effectors. The crystal structures of the non-allosteric PKs from cat and rabbit muscle are known. We have determined the three-dimensional structure of the allosteric type I PK from Escherichia coli, in order to study the mechanism of allosteric regulation. RESULTS: The 2.5 A resolution crystal structure of the unligated type I PK in the inactive T-state shows that each subunit of the homotetrameric enzyme comprises a (beta/alpha)8-barrel domain, a flexible beta-barrel domain and a C-terminal domain. The allosteric and active sites are located at the domain interfaces. Comparison of the T-state E. coli PK with the non-allosteric muscle enzyme, which is thought to adopt a conformation similar to the active R-state, reveals differences in the orientations of the beta-barrel and C-terminal domains of each subunit, which are rotated by 17 degrees and 15 degrees, respectively. Moreover, the relative orientation of the four subunits differs by about 16 degrees in the two enzymes. Highly conserved residues at the subunit interfaces couple these movements to conformational changes in the substrate and allosteric effector binding sites. The subunit rotations observed in the T-state PK induce a shift in loop 6 of the (beta/alpha)8-barrel domain, leading to a distortion of the phosphoenolpyruvate-binding site accounting for the low substrate affinity of the T-state enzyme. CONCLUSIONS: Our results suggest that allosteric control of PK is accomplished through remarkable domain and subunit rotations. On transition from the T- to the R-state all 12 domains of the functional tetramer modify their relative orientations. These concerted motions are the molecular basis of the coupling between the active centre and the allosteric site.

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity.

BACKGROUND: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis. RESULTS: The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme. CONCLUSIONS: The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.

Cross-talk and ammonia channeling between active centers in the unexpected domain arrangement of glutamate synthase.

INTRODUCTION: The complex iron-sulfur flavoprotein glutamate synthase catalyses the reductive synthesis of L-glutamate from 2-oxoglutarate and L-glutamine, a reaction in the plant and bacterial pathway for ammonia assimilation. The enzyme functions through three distinct active centers carrying out L-glutamine hydrolysis, conversion of 2-oxoglutarate into L-glutamate, and electron uptake from an electron donor. RESULTS: The 3.0 A crystal structure of the dimeric 324 kDa core protein of a bacterial glutamate synthase was solved by the MAD method, using the very weak anomalous signal of the two 3Fe-4S clusters present in the asymmetric unit. The 1,472 amino acids of the monomer fold into a four-domain architecture. The two catalytic domains have canonical Ntn-amidotransferase and FMN binding (beta/alpha)8 barrel folds, respectively. The other two domains have an unusual "cut (beta/alpha)8 barrel" topology and an unexpected novel beta-helix structure. Channeling of the ammonia intermediate is brought about by an internal tunnel of 31 A length, which runs from the site of L-glutamine hydrolysis to the site of L-glutamate synthesis. CONCLUSIONS: The outstanding property of glutamate synthase is the ability to coordinate the activity of its various functional sites to avoid wasteful consumption of L-glutamine. The structure reveals two polypeptide segments that connect the catalytic centers and embed the ammonia tunnel, thus being ideally suited to function in interdomain signaling. Depending on the enzyme redox and ligation states, these signal-transducing elements may affect the active site geometry and control ammonia diffusion through a gating mechanism.

Crystal structure of apo-avidin from hen egg-white.

The three-dimensional structure of hen egg-white apo-avidin, crystallized in a tetragonal crystal form, has been refined to a crystallographic R-factor of 0.164 (for the 6390 observed reflections in the 10.0 to 2.8 A resolution range). As in the case of holo-avidin, from which starting atomic co-ordinates were derived, the functional tetramer shows 2-pseudo 22 molecular symmetry. Each promoter is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions, which define the biotin binding pocket in the protomer core. In the absence of biotin the binding site is only partly occupied by water molecules. The structure of the binding site residues, as observed in apo-avidin, is highly complementary to that of the incoming biotin molecule, accounting for prompt and specific recognition. A crystal lattice contact may play a role in stabilizing the conformation of one protein loop, part of the biotin-binding pocket.

Three-dimensional structure of the tetragonal crystal form of egg-white avidin in its functional complex with biotin at 2.7 A resolution.

The three-dimensional structure of hen egg-white avidin, crystallized in a tetragonal crystal form, has been solved at 2.7 A resolution by molecular replacement methods. After refinement the crystallographic R-factor is 16.8%, for the 7255 reflections in the 10.0 to 2.7 A resolution range. The asymmetric unit contains two avidin polypeptide chains (M(r) 2 x 15,600), which build up the functional tetramer through a crystallographic 2-fold axis parallel to the c unit cell direction. The avidin tetramer has almost exact 222 molecular symmetry; the three possible dimers display quite distinct packing interfaces. Each protomer is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions. The avidin binding site within each promoter is located in a deep pocket, at the center of the barrel, displaying both hydrophobic and polar residues for recognition of the tightly bound vitamin. Two Trp residues, Trp70 and Trp97, and Phe79 are in close contact with biotin. Moreover, the binding pocket is partly closed in its outer rim by residue Trp110 of a neighboring subunit. Once bound, biotin is almost completely buried in the protein core, with the exception of the valeryl side-chain carboxylate group which is exposed to solvent, hydrogen bonds to residues Ala39, Thr40 and Ser75, and triggers the formation of a network of hydrogen bonded water molecules. Modeling of synthetic biotin analogues allows us to rationalize functional data available for the binding of these compounds, and to analyze them in terms of biotin recognition mechanism. Hen egg-white avidin shows clear structural homology to streptavidin, from Streptomyces avidinii, but significant deviations can be observed in some regions.

Crystal structure of ferric Aplysia limacina myoglobin at 2 X 0 A resolution.

The three-dimensional structure of ferric myoglobin from the mollusc Aplysia limacina has been refined at 2 X 0 A resolution. The crystallographic R factor, calculated at this stage, is 0 X 194. Despite its high content of apolar residues (both aromatic and aliphatic), Aplysia limacina myoglobin, which contains only one histidine residue (at the proximal position), has a structure that conforms to the common eight-helices globin fold observed in other phyla.

Crystallization of the macromolecular complex transthyretin-retinol-binding protein.

Single crystals of the macromolecular complex transthyretin-retinol-binding protein have been obtained. Transthyretin is a carrier of the hormone thyroxine in plasma whereas retinol-binding protein is the specific transporter of the alcohol form of vitamin A in the same medium. This macromolecular complex is found under physiological conditions in plasma and is believed to play an important physiological role. The complex can be formed in vitro by proteins purified from different species. Our crystals are grown with chicken retinol-binding protein complexed to human transthyretin. They are grown by equilibrium dialysis versus 2.3 M ammonium sulphate, 3% ethylene glycol buffered with 0.1 M succinate (pH 5.5). Their space group is I222 (or I2(1)2(1)2(1)) with unit cell parameters a = 222.4 A, b = 163.4 A and c = 55.5 A. Using a conventional X-ray source, we have collected a complete data set of the crystals to a nominal resolution of 3.1 A.

Structural bases for sulfide recognition in Lucina pectinata hemoglobin I.

The X-ray crystal structure of the sulfide derivative of ferric Lucina pectinata hemoglobin component I (HbI) has been determined at 1.9 A resolution (R-factor 0.186). The heme pocket structural organization of HbI is in keeping with its ligand binding properties. The fast sulfide association rate constant can be related to the presence of Gln(64)E7, as the heme distal residue, together with the protein structural properties in the CD-E distal region. Moreover, the very high sulfide affinity for HbI is reflected by the exceptionally slow ligand dissociation rate. The stabilization of the heme-bound sulfide molecule is achieved through hydrogen bonding to Gln(64)E7, as well as by finely tuned aromatic-electrostatic interactions with the clustered residues Phe(29)B10, Phe(43)CD1 and Phe(68)E11. Such a peculiar arrangement of phenylalanyl residues at the distal ligand binding site has not been observed before in the globin family, and is unique to HbI, a protein functionally devoted to sulfide transport.

Crystallization of an odorant-binding protein from cow nasal mucosa.

The first odorant-binding protein isolated from mammalian nasal mucosa is a dimer of subunits of identical molecular weight (19,000) that specifically binds bell pepper odorants, "green" smelling compounds. The protein can be purified in milligram quantities from tissue extractions by sequential use of a silica based anion exchange column and Mono-P chromatofocussing column. In the presence of the binding compound 2-isobutyl-3-methoxypyrazine and of the organic solvent 2-methyl-2,4-pentanediol (17%, v/v), the protein crystallizes in the monoclinic space group P2(1), with unit cell constants a = 54.3 A, b = 66.7 A, c = 41.5 A, beta = 97.2 degrees. From consideration of the crystal packing densities compatible with its unit cell, it can be concluded that two subunits of 19,000 Mr each are present in the asymmetric unit. The diffraction pattern on still photographs of this crystal form of the protein extends to 2.5 A resolution and allows for a detailed crystallographic investigation.

Aplysia limacina myoglobin. Crystallographic analysis at 1.6 A resolution.

The crystal structure of the ferric form of myoglobin from the mollusc Aplysia limacina has been refined at 1.6 A resolution, by restrained crystallographic refinement methods. The crystallographic R-factor is 0.19. The tertiary structure of the molecule conforms to the common globin fold, consisting of eight alpha-helices. The N-terminal helix A and helix G deviate significantly from linearity. The distal residue is recognized as Val63 (E7), which, however, does not contact the heme directly. Moreover the sixth (distal) co-ordination position of heme iron is not occupied by a water molecule at neutrality, i.e. below the acid-alkaline transition point of A. limacina myoglobin. The heme group sits in its crevice in the conventional orientation and no signs of heme isomerism are evident. The iron atom is 0.26 A out of the porphyrin plane, with a mean Fe-N (porphyrin) distance of 2.01 A. The co-ordination bond to the proximal histidine has a length of 2.05 A, and forms an angle of 4 degrees with the heme normal. A plane containing the imidazole ring of the proximal His intersects the heme at an angle of 29 degrees with the (porphyrin) 4N-2N direction. Inspection of the structure of pH 9.0 indicates that a hydroxyl ion is bound to the Fe sixth co-ordination position.

A new crystalline modification of the copper enzyme ascorbate oxidase.

The copper enzyme ascorbate oxidase, purified from green zucchini squash, has been crystallized at pH 5.4 employing the organic solvent 2-methyl-2,4-pentanediol. The crystals obtained are larger than one millimetre and belong to the space group P2(1)2(1)2, with unit cell parameters; a = 106.7 A, b = 105.1 A, c = 113.5 A. The crystallographic asymmetric unit contains two subunits of the enzyme (Mr = 140,000) and the solvent content of the crystals is 46% (v/v). The diffraction pattern extends to 2.5 A resolution; this crystal form is suitable for a X-ray structural investigation.