X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (Mo^VI) protein has been investigated in solution at 77?K; the Mo K-edge position (20006.4?eV) is consistent with the presence of Mo^VI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71?Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32?Å and two at 2.47?Å, and two O atoms, one at 1.92?Å (considered to be H-bonded to Trp 116) and one at 2.27?Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (Mo^VI) DMSO reductase at 77?K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (Mo^VI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4?eV and 20004.5?eV, respectively; these values, together with the lack of a significant presence of Mo^V in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of Mo^IV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05?Å and one at 2.51?Å, and four S atoms at 2.36?Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69?Å, one at 1.91?Å and one at 2.11?Å, plus four S atoms, two at 2.28?Å and two at 2.37?Å. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to Mo^IV with an Mo-O bond of length 1.92?Å.     

Binding of ruthenium(III) anti-tumor drugs to human lactoferrin probed by high resolution X-ray crystallographic structure analyses

The binding to human lactoferrin of three Ru(III) complexes with anti-tumor activity has been investigated by X-ray crystallography in order to gain insights into how such complexes might be carried during transferrin-mediated delivery to cells. The complexes, HIm[RuIm₂Cl₄], HInd[RuInd₂Cl₄] and (HInd)₂ [RuIndCl₅], where Im?=?imidazole and Ind?=?indazole, were diffused into crystals of apo-lactoferrin (apoLf). X-ray diffraction data were collected to 2.6?Å, 2.2?Å and 2.4?Å respectively. The binding sites for the Ru complexes were determined from difference Fouriers, in comparison with native apoLf; the two indazole-apoLf complexes were also refined crystallographically to final R factors of 0.202 (for 8.0 to 2.3?Å data) and 0.192 (for 8.0 to 2.4?Å data) respectively. Two types of binding site were identified, a high-affinity site at His 253 in the open N-lobe iron-binding cleft of apoLf (and by analogy a similar one at His 597 in the C-lobe), and lower-affinity sites at surface-exposed His residues, primarily His 590 and His 654. The exogenous heterocyclic ligands remain bound to Ru, at least at the His 253 site, and modelling suggests that the nature and number of these ligands may determine whether the closed structure that is required for receptor binding could be formed or not. The results also highlight the importance of His residues for binding such complexes and the value of heavy atom binding studies from crystallographic analyses for identifying non-specific binding sites on proteins.     

Filamentous Bacterial viruses

The filamentous bacterial virus is a simple and well-characterized model system for studying how genetic information is transformed into molecular machines. The viral DNA is a single-stranded circle coding for about 10 proteins. The major viral coat protein is largely α-helical, with about 46 amino acid residues. Several thousand identical copies of this protein in a helical array form a hollow cylindrical tube 1–2μ long, of outer diameter 60 Å and inner diameter 20 Å, with the twisted circular DNA extending down the core of the tube. Before assembly, the viral coat protein spans the cell membrane, and assembly involves extrusion of the coat from the membrane. X-ray fibre diffraction patterns of the Pf 1 species of virus at 4°C, oriented in a strong magnetic field, give three-dimensional data to 4 Å resolution. An electron density map calculated from native virus and a single iodine derivative, using the maximum entropy technique, shows a helix pitch of 5.9 Å. This may indicate a stretched A-helix, or it may indicate a partially 3₁₀ helix conformation, resulting from the fact that the coat protein is an integral membrane protein before assembly, and is still in the hydrophobic environment of other coat proteins after assembly.     

Solution structure of reduced Clostridium pasteurianum rubredoxin

The solution structure of reduced Clostridium pasteurianum rubredoxin (MW 6100) is reported here. The protein is highly paramagnetic, with iron(II) being in the S=2 spin state. The Hβ protons of the ligating cysteines are barely observed, and not specifically assigned. Seventy-six percent of the protons have been assigned and 1267 NOESY peaks (of which 1037 are meaningful) have been observed. Nonselective T ₁ measurements have been measured by recording four nonselective 180°-τ-NOESY at different τ values, and fitting the intensity recoveries to an exponential recovery. Thirty-six metal-proton upper and lower distance constraints have been obtained from the above measurements. The use of such constraints is assessed with respect to spin delocalization on the sulfur donor atoms. The solution structure obtained with the program DYANA has been refined through restrained energy minimization. A final family of 20 conformers is obtained with no distance violations larger than 0.24?Å, and RMSD values to the mean structure of 0.58 and 1.03?Å for backbone and all heavy atoms, respectively (measured on residues 3–53). The structure is compared to the X-ray structure of the oxidized and of the zinc substituted protein, and to the available structures of other rubredoxins. In particular, the comparison with the crystal structure and the solution structure of the Zn derivative of the highly thermostable Pyrococcus furiosus rubredoxin suggested that the relatively low thermal stability of the clostridial rubredoxin may be tentatively ascribed to the loosening of its secondary structure elements. This research is a further achievement at the frontier of solution structure determinations of paramagnetic proteins.     

Preliminary crystallographic study on cytochrome c3 of Desulfovibrio desulfuricans (strain Norway).

Crystals of the quadrihaemic cytochrome c₃ (M_r = 13,000) from Desulfovibrio desulfuricans Norway and of two heavy-atom derivatives have been obtained. X-ray diffraction intensities have been collected down to 3 Å resolution for the native protein crystals and the mercury derivative.     

EXAFS and Mössbauer characterization of the Diiron(III) site in stearoyl-acyl carrier protein Δ9– desaturase

Stearoyl-acyl carrier protein (ACP) Δ⁹-desaturase (Δ9D) from the castor plant is the best characterized soluble acyl-ACP desaturase. This enzyme utilizes a diiron center to catalyze the O₂- and NADPH-dependent introduction of a cis double bond between carbons 9 and 10 of stearoyl-ACP, yielding oleoyl-ACP. In the present study, we have used X-ray absorption spectroscopy to provide the first metrical information for the diferric oxidation state. These studies reveal distinct diiron clusters that have Fe-Fe distances of either 3.12 or 3.41?Å. The species having the 3.12?Å Fe-Fe distance also exhibits a 1.8?Å Fe-O bond and is thus proposed to represent Δ9D molecules containing a (μ-oxo)bis(μ-carboxylato)diiron(III) cluster. The species having the 3.41?Å Fe-Fe distance exhibits no short Fe-O bond, and thus likely represents Δ9D molecules containing a (μ-hydroxo)diiron(III) cluster. Mössbauer studies of the extended X-ray absorption fine structure (EXAFS) samples revealed three quadrupole doublets (ΔE _Q(1)=1.53?mm/s, 72%;ΔE _Q(2)=0.72?mm/s, 21%;ΔE _Q(3)=2.20?mm/s, 7%) that originate from three distinct dinuclear clusters. From analysis of spectral intensities and by comparison with previous studies of (μ-oxo)- and (μ-hydroxo)diiron(III) clusters in both model complexes and proteins, doublet 1, the Mössbauer majority species, is likely associated with the EXAFS majority species having a 3.12?Å Fe-Fe separation and a 1.8?Å Fe-μ-oxo bond, while doublet 2 likely results from one iron site (or both) of a cluster associated with the EXAFS species having a 3.41?Å Fe-Fe separation. The presence of multiple diiron center conformations in diferric Δ9D may reflect the necessity for the active site to allow access of the substrate stearoyl-ACP (～9?kDa) during desaturation catalysis.     

Structure Determination of Functional Membrane Proteins using Small-Angle Neutron Scattering (SANS) with Small, Mixed-Lipid Liposomes: Native Beef Heart Mitochondrial Cytochrome c Oxidase Forms Dimers

The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

A low-resolution model of crystalline methionyl-transfer RNA synthetase from Escherichia coli

When submitted to a controlled proteolysis by trypsin, native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) yields a well-defined fragment of molecular weight 64,000 composed of one single polypeptide chain. This fragment retains full specificity towards methionine and tRNA^met, and has unimpaired activity in both the activation reaction and aminoacyl-tRNA formation. Crystals of this active fragment have been studied by X-ray crystallography and, using two isomorphous heavy-atom derivatives, a 4 Å electron density map has been calculated.The molecule appears as an elongated ellipsoid of overall dimensions 90 Å × 43 Å × 43 Å. It is clearly built of two parts separated by a large cleft. The volume of one of these “domains” is approximately twice that of the other; these results are consistent with our present knowledge of the chemistry of the protein.     

Characterization of a modified nitrogenase Fe protein from Klebsiella pneumoniae in which the 4Fe4S cluster has been replaced by a 4Fe4Se cluster

The Azotobacter vinelandii nifS gene product has been used with selenocysteine to reconstitute Klebsiella pneumoniae nitrogenase Fe protein. Chemical analysis and extended X-ray absorption fine structure (EXAFS) spectroscopy show that the 4Fe4S cluster present in the native protein is replaced by a 4Fe4Se cluster. As well, EXAFS spectroscopy shows that the bond lengths to the cysteine thiolate ligands shrink by 0.05 Å (from 2.28 to 2.23 Å) upon reduction, whereas the Fe–Fe distance is essentially unchanged. Thus, the core of the 4Fe4Se cluster remains essentially static on reduction, whilst the external cysteine thiolate ligands are pulled in towards the cluster. Compared with native (S)–Fe protein, the (Se)–Fe protein has a 20-fold increased rate of MgATP-induced Fe chelation, a sixfold decreased specific activity for acetylene reduction, a fivefold decreased rate of MgATP-dependent electron transfer from (Se)–Fe protein to MoFe protein, and a fourfold increase in the ATP to 2e ^? ratio. The high ATP to 2e ^? ratio and decreased specific activity are consistent with a lower rate of dissociation of oxidized (Se)–Fe protein from reduced MoFe protein. Thus, the relatively small adjustments in the Fe protein structure necessary to accommodate the 4Fe4Se cluster are transmitted both to adjacent residues that dock at the surface of the MoFe protein and to the ATP hydrolysis sites located approximately 19 Å away.     

Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus: crystal structure of the oxidised enzyme at 1.82-Å resolution and the dithionite-reduced enzyme at 2.8-Å resolution

The 1.82-Å X-ray crystal structure of the oxidised (Mo^(VI)) form of the enzyme dimethylsulfoxide reductase (DMSOR) isolated from Rhodobacter capsulatus is presented. The structure has been determined by building a partial model into a multiple isomorphous replacement map and fitting the crystal structure of DMSOR from Rhodobacter sphaeroides to the partial model. The enzyme structure has been refined, at 1.82-Å resolution, to an R factor of 14.8% (R _free?=?18.4%). The molybdenum is coordinated by seven ligands: four dithiolene sulfurs, Oγ of Ser147 and two oxo groups. The four sulfur ligands, at a metal-sulfur distance of 2.4?Å or 2.5?Å, are contributed by the two molybdopterin guanine dinucleotide (MGD) cofactors. The coordination sphere of the molybdenum is different from that in previously reported structures of DMSOR from R. sphaeroides and R. capsulatus. The 2.8-Å structure of DMSOR, reduced by addition of sodium dithionite, is also described and differs from the structure of the oxidised enzyme by the removal of a single oxo ligand from the molybdenum coordination sphere. A structure, at 2.5-Å resolution, has also been obtained from crystals soaked in mother liquor buffered at pH?7.0. No differences are observed in the structure at pH?7 when compared with the native crystal structure at pH?5.5.     

Monte carlo simulations of protein folding. II. Application to protein A,ROP, and crambin

The hierarchy of lattice Monte Carlo models described in the accompanying paper (Kolinski, A., Skolnick, J. Monte Carlo simulations of protein folding. I. Lattice model and interaction scheme. Proteins 18:338–352, 1994) is applied to the simulation of protein folding and the prediction of 3-dimensional structure. Using sequence information alone, three proteins have been successfully folded: the B domain of staphylococcal protein A, a 120 residue, monomeric version of ROP dimer, and crambin. Starting from a random expanded conformation, the model proteins fold along relatively well-defined folding pathways. These involve a collection of early intermediates, which are followed by the final (and rate-determining) transition from compact intermediates closely resembling the molten globule state to the native-like state. The predicted structures are rather unique, with native-like packing of the side chains. The accuracy of the predicted native conformations is better than those obtained in previous folding simulations. The best (but by no means atypical) folds of protein A have a coordinate rms of 2.25 Å from the native Cα trace, and the best coordinate rms from crambin is 3.18 Å. For ROP monomer, the lowest coordinate rms from equivalent Cαs of ROP dimer is 3.65 Å. Thus, for two simple helical proteins and a small α/β protein, the ability to predict protein structure from sequence has been demonstrated. © 1994 John Wiley & Sons, Inc.     

Structural characterization of the mononuclear iron site in Pseudomonas cepacia phthalate DB01 dioxygenase using X-ray absorption spectroscopy

Phthalate dioxygenase (PDO) from Pseudomonas cepacia contains a Rieske-like [2Fe-2S] cluster and a mononuclear non-heme Fe(II) site. The mononuclear iron can be replaced by a variety of divalent metal ions, although only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuclear iron site and to follow the structural changes in this site as a function both of Rieske site oxidation state and of phthalate binding. Data for the mononuclear site have been measured directly for PDO substituted with Co or Zn in the mononuclear site, and by difference for the native 3-Fe protein. The mononuclear site was modeled well by low Z-ligation (oxygen or nitrogen) and showed no evidence for high-Z ligands (e.g., sulfur). The relatively short average first shell bond lengths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands. With Zn in the mononuclear site, the average bond length (2.00?Å) suggests a 5-coordinate site under all conditions. In contrast, the Co- or Fe-containing mononuclear site appeared to be 6-coordinate and changed to 5-coordinate when substrate was bound, since the first shell bond length changed from 2.08 to 2.02?Å (Co) or 2.10 to 2.06?Å (Fe). The implications of these findings for the PDO mechanism are discussed.     

EXAFS studies on the PN and POX states of the P-clusters in nitrogenase

The MoFe protein of nitrogenase is an α₂β₂ tetramer that contains two each of two different types of metal centers, the FeMo-cofactor and the P-clusters. The function of the P-clusters is believed to be to accept electrons from the Fe protein of nitrogenase and to donate them to the FeMo-cofactor. We have studied the P-clusters of Azotobacter vinelandii nitrogenase in both the P^N and P^OX states utilizing Fe K-edge X-ray absorption spectroscopy. Since the MoFe holoprotein contains the seven iron FeMo-cofactor centers in addition to P-clusters, we have utilized a FeMo-cofactor-deficient MoFe protein synthesized by the Δnif H strain DJ54. That MoFe protein is an α₂β₂ tetramer that contains P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the P^OX state. Several important results have emerged from our XAS studies. The first shell Fe-S coordination shows the same average Fe-S distance (2.26?Å) in both states. The second coordination shells could only be well fit using two different Fe-Fe contributions. In both states, short Fe-Fe components with distances of 2.57?Å and 2.42?Å for the P^N and P^OX states, respectively, were required to complement longer 2.75?Å and 2.70?Å distances. Understanding of the P-cluster structure is essential if we are to make advances in understanding the role of the P-clusters and their participation in electron transfer through the nitrogenase system.     

A study of protein structure changes during hydration by diffuse X-ray scattering: II. Fourier transform analysis of diffuse X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of the angular dependences of diffuse X-ray scattering intensities for the following proteins with different hydration degrees: water-soluble α-protein myoglobin, water-soluble (α + β) protein lysozyme, and transmembrane proteins from the photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides and Blastochlorii (Rhodopseudomonas) viridis. The results of Fourier transform analysis of X-ray scattering intensities give quantitative characteristics of the mechanism underlying the influence of water on the formation of biological macromolecules. On the one hand, water loosens the network of hydrogen bonds, which results in a considerable conformational mobility in the molecules of lysozyme and myoglobin and the reaction centers. On the other hand, water stabilizes and orders the protein globule. A strict correlation was found between the shift of the “first” maximum of the radial distribution function, loosening of the intraglobular hydrogen bonds, increase in the intramolecular mobility, and appearance of pronounced functional activity in macromolecules. The pattern of behavior of the first maximum in the transmembrane proteins of the reaction center was similar to that observed for the water-soluble proteins. However, the first maximum reached the limiting value of 2.9 Å at a considerably lower hydration degree compared with the water-soluble proteins. A quick transition of the protein complex of the reaction center to its native state is due to the fact that the dehydrated conformation of this complex is very close to the native conformation. Comparison of the radial distribution function for water, water-soluble proteins, and transmembrane proteins suggests a quantitative conclusion that water is the least densely packed and ordered system, the water-soluble proteins are more densely packed than water, and the transmembrane proteins are the most densely packed and ordered system.     

Neocarzinostatin: an antitumor protein a preliminary x-ray diffraction study.

The antitumor protein, neocarzinostatin, has been crystallized and examined by X-ray diffraction. Crystals of this globular protein are of space group P2₁2₁2₁ with cell parameters a = 27.4Å, b = 33.9Åand c = 102.0Å. There is one molecule of approximately 27Ådiameter per asymmetric unit. Crystals soaked in a K₂HgI₄ solution give diffraction patterns significantly different from native crystal diffraction patterns.     

Changes in the x-ray solution scattering of aspartate transcarbamylase following the allosteric transition.

Aspartate transcarbamylase (Escherichia coli) has been studied by X-ray solution scattering in the s range 0.002 to 0.06 Å^?1. The spectra display sharp maxima and minima whose positions and amplitudes show considerable changes upon ligation with the transition state analogue N-(phosphonacetyl)-l-aspartate. The magnitude of the change in diffraction pattern is so large that X-ray solution scattering should be a useful technique for studying the proportions of different quaternary forms in solutions of this enzyme. In particular, the kinetics of the allosteric transition appear to be within the reach of X-ray diffraction experiments.Some structural parameters of the allosteric transition were obtained from the diffraction patterns. The radius of gyration of the native enzyme is 45.9 ± 0.5 Å, and after ligation it increases to 48.4 ± 1.0 Å. At the same time, the peak of the pair distribution function is shifted from 58 Å to 63 Å. These changes indicate that the molecule swells after the allosteric transition to the R form. However, the maximum distance (from the pair distribution function) does not increase after ligation, and may even decrease slightly. Some probable subunit movements during allosteric activation are discussed.     

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn²⁺. The EPR spectrum of Mn²⁺–activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5?(±0.1)?Å. In the X-ray absorption spectrum of Mn²⁺–activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k≤12?Å^–1 by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15?Å, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5?Å is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn²⁺–activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.