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E N Baker

Publications and source records attributed to E N Baker.

At least 91 records · Page 5Linked to original sources
PubMed1995

Crystallization and preliminary X-ray diffraction studies of arginase from a thermophilic organism Bacillus caldevelox.

A thermostable hexameric arginase purified from the extreme thermophile Bacillus caldevelox has been crystallized from Hepes buffer at pH 7.5 in the presence of 12% polyethylene glycol 4000 and 10% 2-propanol, and from cacodylate buffer at pH 7.2 in the presence of 15% 2-propanol and sodium citrate. The latter crystals are more suitable for X-ray diffraction analysis. The crystals are in the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions a = 156.3, b = 148.0 and c = 85.4 A. The asymmetric unit contains one hexamer (approximate molecular mass 183 kDa) and has a solvent content of approximately 54%. The crystals diffract to 2.8 A resolution.

Journal Article↗
PubMed1995

1.8 A crystal structure of the C-terminal domain of rabbit serum haemopexin.

BACKGROUND: Haemopexin is a serum glycoprotein that binds haem reversibly and delivers it to the liver where it is taken up by receptor-mediated endocytosis. Haemopexin has two homologous domains, each having a characteristic fourfold internal sequence repeat. Haemopexin-type domains are also found in other proteins, including the serum adhesion protein vitronectin and various collagenases, in which they mediate protein-protein interactions. RESULTS: We have determined the crystal structure of the C-terminal domain of haemopexin at 1.8 A resolution. The domain is folded into four beta-leaflet modules, arranged in succession around a central pseudo-fourfold axis. A funnel-shaped tunnel through the centre of this disc-shaped domain serves as an ion-binding site. CONCLUSIONS: A model for haem binding by haemopexin is proposed, utilizing an anion-binding site at the wider end of the central tunnel, together with an associated cleft. This parallels the active-site location in other beta-propeller structures. The capacity to bind both cations and anions, together with the disc shape of the domain, suggests that such domains may be used widely for macromolecular recognition.

Amino Acid Sequence↗
PubMed1995

Use of iron anomalous scattering with multiple models and data sets to identify and refine a weak molecular replacement solution: structure analysis of cytochrome c' from two bacterial species.

The structure of cytochrome c' from two bacterial species, Alcaligenes sp and Alcaligenes denitrificans, have been determined from X-ray diffraction data to 3.0 A resolution using the anomalous scattering of the single Fe atom in each to identify and refine a weak molecular-replacement solution. Molecular-replacement studies, with the program AMORE, used two isomorphous data sets (from the two species), two independent search models (the cytochromes c' from Rhodospirillum molischianum and Rhodospirillum rubrum), both with and without side chains, and two different resolution ranges (10.0-4.0 and 15.0-3.5A) to generate a large number of potential solutions. No single solution stood out and none appeared consistently. The Fe-atom position in each structure was then determined from its anomalous-scattering contribution and all molecular- replacement solutions were discarded which did not (i) place the Fe atom correctly and (ii) orient the molecule such that a crystallographic twofold axis generated a dimer like those of the two search models. Finally, electron-density maps phased solely by the Fe-atom anomalous scattering were calculated. As these were combined and subjected to solvent flattening and histogram matching (with the program SQUASH), correlation with the remaining molecular-replacement solutions identified one as correct and enabled it to be improved and subjected to preliminary refinement. The correctness of the solution is confirmed by parallel isomorphous-replacement studies.

Journal Article↗
PubMed1994

The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum.

BACKGROUND: Peptide:N-glycosidase F (PNGase F) is an enzyme that catalyzes the complete removal of N-linked oligosaccharide chains from glycoproteins. Often called an endoglycosidase, it is more correctly termed an amidase or glycosylasparaginase as cleavage is at the asparagine-sugar amide linkage. The enzyme is widely used in structure-function studies of glycoproteins. RESULTS: We have determined the crystal structure of PNGase F at 1.8 A resolution. The protein is folded into two domains, each with an eight-stranded antiparallel beta jelly roll configuration similar to many viral capsid proteins and also found, in expanded form, in lectins and several glucanases. Two potential active site regions have been identified, both in the interdomain region and shaped by prominent loops from one domain. Exposed aromatic residues are a feature of one site. CONCLUSIONS: The finding that PNGase F is based on two jelly roll domains suggests parallels with lectins and other carbohydrate-binding proteins. These proteins either bind sugars on the concave face of the beta-sandwich structure (aided by loops) or amongst the loops themselves. Further analysis of the function and identification of the catalytic site should lead to an understanding of both the specificity of PNGase F and possibly also the recognition processes that identify glycosylation sites on proteins.

Amidohydrolases↗
PubMed1994

Purification and crystallization of the endoglycosidase PNGase F, a peptide:N-glycosidase from Flavobacterium meningosepticum.

The endoglycosidase peptide: N-glycosidase, secreted by the Gram-negative bacterium Flavobacterium meningosepticum (PNGase F), has been isolated, purified to homogeneity and crystallized from polyethylene glycol solutions using vapour diffusion and seeding techniques. The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 85.07 A, b = 85.14 A, c = 48.50 A, and are suitable for high resolution X-ray structure analysis.

Amidohydrolases↗
PubMed1994

Crystallization and preliminary X-ray diffraction studies on cytosolic (class 1) aldehyde dehydrogenase from sheep liver.

The cytosolic (Class 1) aldehyde dehydrogenase (AlDH) from sheep liver has been crystallized in a form suitable for X-ray diffraction studies. The crystals, grown by vapour diffusion using 6.5 to 7.5% methoxypolyethylene glycol 5000 as precipitant, at pH 6.5, are orthorhombic with cell dimensions a = 80.7, b = 92.5, c = 151.6 A, space-group P2(1)2(1)2(1), and one dimer in the asymmetric unit. The crystals diffract to at least 2.8 A resolution. Although unmodified AlDH crystallized readily, a key factor in obtaining diffraction-quality crystals was the covalent attachment of an active site reporter group, provided by 3,4-dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one.

Aldehyde Dehydrogenase↗
PubMed1994

Enzymatic deglycosylation as a tool for crystallization of mammalian binding proteins.

Enzymatic deglycosylation has been used in attempts to crystallize several glycoproteins with the aim of overcoming the problems resulting from heterogeneity and flexibility of the attached glycan chains. An endoglycosidase preparation from Flavobacterium meningosepticum, comprising the enzymes endo F and PNGase-F, was used in experiments on the mammalian binding proteins lactoferrin and haemopexin. Significant differences were found in the susceptibility of different proteins to deglycosylation. For human lactoferrin (Lf) and its recombinant N-terminal half-molecule (Lf(N)), deglycosylation was rapid and complete, and was essential for obtaining high-quality crystals of both apo-Lf and Lf(N); for bovine Lf, however, complete deglycosylation did not occur. Similarly, for rabbit haemopexin the carbohydrate chain on the C-terminal domain was easily removed, but the three chains on the N-terminal domain proved more resistant and their removal led to some fragmentation of the protein. Nevertheless, this approach provided the only means of crystallizing the C-terminal domain and is likely to be useful for other glycoproteins.

Journal Article↗
PubMed1994

Search designs for protein crystallization based on orthogonal arrays.

In protein crystallography, the initial experimental problem is the identification of physical and chemical conditions that will support nucleation and crystal growth. Ideally, experiments to search for such conditions would be based on a full-factorial structure, with variation in the temperature and solution composition. However, consideration of even a moderate number of possibilities for the composition of the system will result in factorial experiments which may be prohibitively large. In this paper it is proposed that search experiments for protein crystallization might be based on orthogonal arrays. These are subsets of full-factorial experiments which possess a great deal of symmetry, such that a uniform distribution of points throughout the experimental region is preserved. Such experiments have reasonable size, explore the proposed experimental region in a systematic fashion, and form a logical basis for a sequential approach to the search for crystallization conditions. Examples of such initial search experiments are given, and their application to some recent protein crystallization problems in this laboratory is described briefly. The relationship of this approach to other protein crystallization search procedures is also discussed.

Journal Article↗
PubMed1994

Metal substitution in a blue-copper protein: the crystal structure of cadmium-azurin at 1.8 A resolution.

Crystals of cadmium-substituted azurin have been prepared by diffusing Cd(II) into crystals of apo-azurin grown previously and their structure has been determined at high resolution by X-ray crystallography. Data to 1.8 A resolution were collected by Weissenberg photography (with image plates) using synchrotron radiation. These data were combined with a 2.2 A diffractometer data set to give 90% coverage to 1.8 A. An initial model was derived from the isomorphous Cu(II)-azurin structure, and the cadmium and ligand positions added from 'omit' maps. Refinement was by restrained least squares (program PROLSQ), to a final R value of 0.168 for all data in the range 10.0-1.8 A (23 349 reflections). The final model of 1954 protein atoms, two Cd(II) ions (occupancy 0.75), four SO(4)(2-) ions and 239 water molecules has r.m.s. deviations of 0.015, 0.045 and 0.013 A from standard bond lengths, angle distances and planar groups. The protein structure is essentially the same as that of Cu(II)-azurin, with an r.m.s. deviation of 0.18 A for 97% of main-chain atoms after superposition of the two structures. The Cd atom is within 0.2 A of the equivalent copper position, displaced slightly away from the axial Met ligand towards the carbonyl O atom of Gly45. The latter has also moved slightly towards the metal, by a rotation of the peptide unit, to give a Cd-O bond of 2.76 A. The Cd-S(Cys) bond is lengthened to 2.39 A. The coordination geometry is slightly more tetrahedral than for Cu(II), and the cadmium-oxygen interaction is consistent with the presence of an oxygen ligand in the coordination sphere of stellacyanin.

Journal Article↗
PubMed1994

Structure of copper- and oxalate-substituted human lactoferrin at 2.0 A resolution.

The three-dimensional structure of human dicupric monooxalate lactoferrin, Cu(2)oxLf, has been determined to 2.0 A resolution, using X-ray diffraction data collected by diffractometry to 2.5 A resolution, and oscillation photography on a synchrotron source to 2.0 A resolution. Difference electron-density maps calculated between Cu(2)oxLf and both dicupric lactoferrin, Cu(2)Lf, and diferric lactoferrin, Fe(2)Lf, showed that the oxalate had replaced a carbonate in the C-terminal binding site, and that, relative to Cu(2)Lf, there were no significant differences in the N-terminal site. The structure was then refined crystallographically by restrained least-squares methods. The final model, in which the r.m.s. deviation in bond distances is 0.017 A, contains 5314 protein atoms (691 residues), two Cu(2+) ions, one bicarbonate ion, one oxalate ion, 325 solvent molecules and one sugar residue. The crystallographic R factor of 0.193 is for 46 134 reflections in the range 8.0 to 2.0 A resolution. The oxalate ion is coordinated to copper in a 1,2-bidentate fashion, and the added bulk of the anion results in the rearrangement of the side chains of nearby arginine and tyrosine residues. No other major alterations in the molecule can be observed, the overall protein structure being the same as that for Cu(2)Lf and Fe(2)Lf.

Journal Article↗
PubMed1994

Three-dimensional structure of lactoferrin in various functional states.

The three-dimensional structures of various forms of lactoferrin, determined by high resolution crystallographic studies, have been compared in order to determine the relationship between structure and biological function. These comparisons include human apo and diferric lactoferrins, metal and anion substituted lactoferrins, the N-terminal half molecule of human lactoferrin, and bovine diferric lactoferrin. The structures themselves define the nature and location of the iron binding sites and allow anti-bacterial and putative receptor-binding regions to be mapped on to the molecular surface. The structural comparisons show that small internal adjustments can allow the accommodation of different metals and anions without altering the overall molecular structure, whereas large-scale conformational changes are associated with metal binding and release, and smaller, but significant, movements accompany species variations. The results also focus on differences in flexibility between the two lobes, and on the importance of interactions in the inter-lobe region in modulating iron release from the N-lobe and in possibly enabling binding at one site to be signalled to the other.

Animals↗
PubMed1994

Synergism and substitution in the lactoferrins.

The anion binding properties of human lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, highlight differences between the two sites, and in the anion binding behaviour when different metals are bound. Carbonate, oxalate and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf although it does for the bovine analogue. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+. The anion sites as determined from the crystal structures of Fe2(CO3)2Lf, Fe2(C2O4)2Lf, Cu2(CO3)2Lf, and Cu2(CO3)(C2O4)Lf have been compared. Both the carbonate and oxalate ions bind in bidentate fashion to the metal, except that the carbonate ion in the N-lobe site of dicupric lactoferrin is monodentate. The hybrid copper lactoferrin complex shows that the oxalate ion binds preferentially in the C-lobe site in a bidentate mode. A series of complexes containing the synergistic anion O,N-chelates with increasing substitution on the N atom (glycinate, iminodiacetate and nitrilotriacetate) have been prepared with iron bovine lactoferrin for comparison with the O,O-chelate oxalate. Overall these observations lead to a generalised model for synergistic anion binding by transferrins and allow comparisons to be made with nonsynergistic anions such as citrate and succinate.

Animals↗
PubMed1993

Domain closure in lactoferrin. Two hinges produce a see-saw motion between alternative close-packed interfaces.

Lactoferrin is an iron transport protein. Upon binding iron, the two domains in the N-terminal half of the molecule move together. Previous work has shown that this domain closure involves two hinges. Using the newly refined structure of the open form, the structural mechanism underlying this motion is analyzed here in detail. Upon closure the domains rotate 54 degrees essentially as rigid bodies. The axis of rotation passes through the two beta-strands linking the domains. These strands contain hinges in the sense that three large torsion angle changes are responsible for the bulk of the motion while smaller torsion angle changes in neighboring residues are responsible for the remainder of the motion. The rotation axes of these three torsion angle changes are nearly parallel to the axis of the overall 54 degrees rotation, so the local motion in the hinges can be directly related to the overall motion. A crucial feature of the hinge residues is that they have very few packing constraints on their main-chain atoms. The domains make different packing contacts with each other in the open and closed forms. These contacts form two interdomain interfaces arranged on either side of the hinges. Pivoting about the hinges produces a see-saw motion between the two interfaces. That is, when the domains close down, residues in the interface on one side of the hinges become buried and close-packed and residues on the other side become exposed. The situation is reversed when the domains open up. Lactoferrin provides a particularly clear example of the general features of hinged domain motion. It is compared to other instances of hinged domain closure and contrasted with instances of shear domain closure, where the overall motion is a summation of many small sliding motions between close-packed segments of polypeptide.

Amino Acid Sequence↗