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PubMed2003

Monoclonal antibodies for the structural analysis of the Na+/H+ antiporter NhaA from Escherichia coli.

Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.

Antibodies, Monoclonal↗
PubMed2002

X-ray structure analysis of the sodium salt of beijeran.

The three-dimensional structure of the sodium salt of beijeran has been determined by X-ray fiber diffraction analysis. The acidic polysaccharide forms an extended twofold helix. Two chains are nestled tightly in a monoclinic unit cell of dimensions a=12.72, b=11.41, c (fiber axis)=24.62 A and gamma=123.7 degree in an antiparallel fashion. In the crystalline lattice, helices are stacked tightly to form a thick sheet along the vertical plane passing through the short diagonal of the basal net. Adjacent sheets associate via a network of sodium ions and water molecules embedded between them. The morphology of sodium beijeran in the solid state is consistent with its observed rheological properties.

Azotobacter↗
PubMed1991

Crystal structure analysis of the B-DNA dodecamer CGTGAATTCACG.

The crystal structure of the DNA dodecamer C-G-T-G-A-A-T-T-C-A-C-G has been determined at a resolution of 2.5 A, with a final R factor of 15.8% for 1475 nonzero reflections measured at 0 degrees C. The structure is isomorphous with that of the Drew dodecamer, with the space group P2(1)2(1)2(1) and cell dimensions of a = 24.94 A, b = 40.78 A, and c = 66.13 A. The asymmetric unit contains all 12 base pairs of the B-DNA double helix and 36 water molecules. The structure of C-G-T-G-A-A-T-T-C-A-C-G is very similar to that of C-G-C-G-A-A-T-T-C-G-C-G, with no major alterations in helix parameters. Water peaks in the refined structure appear to represent a selection of peaks that were observed in the Drew dodecamer. The minor-groove spine of hydration at 2.5 A is fragmentary, but as Narendra et al. (1991) [Biochemistry (following paper in this issue)] have observed, lowering the temperature leads to a more complete representation of the spine.

Base Sequence↗
PubMed2001

A surface-associated retinol- and fatty acid-binding protein (Gp-FAR-1) from the potato cyst nematode Globodera pallida: lipid binding activities, structural analysis and expression pattern.

Parasitic nematodes produce at least two structurally novel classes of small helix-rich retinol- and fatty-acid-binding proteins that have no counterparts in their plant or animal hosts and thus represent potential targets for new nematicides. Here we describe a protein (Gp-FAR-1) from the plant-parasitic nematode Globodera pallida, which is a member of the nematode-specific fatty-acid- and retinol-binding (FAR) family of proteins but localizes to the surface of this species, placing it in a strategic position for interaction with the host. Recombinant Gp-FAR-1 was found to bind retinol, cis-parinaric acid and the fluorophore-tagged lipids 11-(dansylamino)undecanoic acid and dansyl-D,L-alpha-amino-octanoic acid. The fluorescence emission characteristics of the dansylated analogues indicated that the entire ligand enters the binding cavity. Fluorescence competition experiments showed that Gp-FAR-1 binds fatty acids in the range C(11) to C(24), with optimal binding at C(15). Intrinsic fluorescence analysis of a mutant protein into which a tryptophan residue had been inserted supported computer-based predictions of the position of this residue at the protein's interior and possibly also at the binding site. Of direct relevance to plant defence systems was the observation that Gp-FAR-1 binds two lipids (linolenic and linoleic acids) that are precursors of plant defence compounds and the jasmonic acid signalling pathway. Moreover, Gp-FAR-1 was found to inhibit the lipoxygenase-mediated modification of these substrates in vitro. Thus not only does Gp-FAR-1 function as a broad-spectrum retinol- and fatty-acid-binding protein, the results are consistent with the idea that Gp-FAR-1 is involved in the evasion of primary host plant defence systems.

Amino Acid Sequence↗
PubMed1993

Nucleic acid structure analysis: a users guide to a collection of new analysis programs.

Common nomenclature describing the geometry of nucleic acid structures was established at a 1988 EMBO Workshop on DNA Curvature and Bending (Diekmann, S. (1988) J. Mol. Biol. 208, 787-791; Diekmann, S. (1989) The EMBO Journal 8, 1-4; Sarma, R.H. (1988) J. Biomol. Structure & Dynamics 6, 391-395; Dickerson, R.E. (1989) J. Biomol. Structure & Dynamics 6, 627-634; Dickerson, R.E. et al. (1989) Nuc. Acids Res. 17, 1979-1803). We have subsequently developed and incorporated sophisticated mathematics in a computer program to calculate the parameters described by the guidelines. The program calculates all the local parameters relating complementary bases and neighboring base and base pairs in both Cartesian and helical coordinate frames. In addition, the main mathematical property requested by the EMBO guidelines--that the magnitude of the parameters be independent of strand or direction of measurement--is accomplished without the use of a midway coordinate frame for the rotational parameters. The mathematics preserve the physical intuition used in defining the parameters; in particular, the rotational parameters are true rotations based on a simple physical model (rotation at constant angular velocity for a unit amount of time), not Euler angles or angles between vectors and planes as is the case with other approaches. As a result, the mathematical equations are symmetric with the property that a 5 degrees tilt is the same as a 5 degrees roll or a 5 degrees twist, except that the rotations take place about different axes. In other approaches, a 5 degrees tilt can mean a different amount of net rotation from a 5 degrees roll or a 5 degrees twist. In addition, a great deal of flexibility is built into the program so that the user has control over the analysis, including the input format, the coordinate frame used for the base pairing relationship, the point about which the rotations are performed, and which geometric relationships are analyzed. While there is a great deal of flexibility, the program is easy to use. Interactive queries and user accessible files make the options in the program very convenient to tailor to individual needs. In addition, there is also a program that calculates bond lengths, valence angles, and torsion angles along the nucleic acid backbone, and within the sugar and base rings. Another program 'learns' the identities of the bond lengths, valence angles, and torsion angles that the user would like to determine.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1992

Three-dimensional structure of acylphosphatase. Refinement and structure analysis.

We report here the complete determination of the solution structure of acylphosphatase, a small enzyme that catalyses the hydrolysis of organic acylphosphates, as determined by distance geometry methods based on nuclear magnetic resonance information. A non-standard strategy for the distance geometry calculations was used and is described here some detail. The five best structures were then refined by restrained energy minimization and molecular dynamics in order to explore the conformational space consistent with the experimental data. We address the question of whether the solution structure of acylphosphatase follows the general principles of protein structure, i.e. those learned from analysing crystal structures. Static and dynamic features are discussed in detail. An uncommon beta-alpha-beta motif, so far found only in procarboxypeptidase B and in an RNA-binding protein, is present in acylphosphatase.

Acid Anhydride Hydrolases↗
PubMed1997

Structural analysis of the lipopolysaccharide from Vibrio cholerae serotype O22.

The structure of the lipopolysaccharide (LPS) from Vibrio cholerae serogroup O22 was elucidated. The LPS was subjected to a variety of degradative procedures, and the structures of the purified products were established by monosaccharide and methylation analyses, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry. The following structure for the complete LPS molecule was determined on the basis of the combined data from these experiments. [formula: see text] The elucidation of this structure provided a chemical basis for the serological cross-reactions observed between this strain and V. cholerae serogroup O139.

Carbohydrate Conformation↗
PubMed1991

Three-dimensional structural analysis of the group B polysaccharide of Neisseria meningitidis 6275 by two-dimensional NMR: the polysaccharide is suggested to exist in helical conformations in solution.

The solution conformations of the group B polysaccharide of Neisseria meningitidis were analyzed by DQF-COSY and pure absorption 2D NOE NMR with three mixing times. The pyranose ring of the sialic acid residue was found to be in the 2C5 conformation. The DQF-COSY analysis indicated that the orientations of H6 and H7 and of H7 and H8 are both gauche. In order to overcome the difficulties in analyzing the NOE data due to the two sets of proton overlaps, molecular modeling of alpha-2,8-linked sialic acid oligomers was carried out to investigate possible conformers, and theoretical NOE calculations were performed by using CORMA (complete relaxation matrix analysis). Our analysis suggests that the polysaccharide adopts helical structures for which the phi (defined by O6-C2-O8-C8) and psi (C2-O8-C8-C7) angles are in the following ranges: phi -60 to 0 degrees, psi 115-175 degrees or phi 90-120 degrees, psi 55-175 degrees. The weak affinity of anti-B antibodies for smaller alpha-2,8-linked oligosaccharides may be due to the fact that such oligomers are more flexible and may not form an ordered structure as the poly(sialic acid) does.

Antigens, Bacterial↗
PubMed1998

Structural analysis of the amplified IFN-beta and DHFR genes in a Chinese hamster ovary cell line using multicolour FISH analysis.

Multicolour FISH was used to get insight into the structural arrangement of a homogeneously staining region which bears the co-transfected and subsequently co-amplified IFN-beta and DHFR genes in a CHO cell line. On metaphase chromosomes an arrangement of multiple bands with regular spacing is revealed. On extended chromatin fibres a cluster of directly repeated and interspersed IFN-beta and DHFR genes is visible. Up to three clusters were found arranged in tandem. The different chromosomal mechanisms leading to gene amplification are discussed.

Animals↗
PubMed2001

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.

Carbohydrate Conformation↗
PubMed1999

Single-crystal structure analysis of a novel aryl phosphinate diglycidyl ether.

The crystal structure of 10-[2,5-bis(2,3-epoxy-1-propoxy)phenyl]-9-oxa-10-phosphaphenanthren-10-one has been studied by single-crystal X-ray diffraction. The unit cell of C(24)H(21)O(6)P, M(r) = 436.4, is triclinic, P1;, with a = 8.507 (3), b = 10.613 (4), c = 12.457 (3) Å, alpha = 80.05 (3), beta = 71.38 (2), gamma = 76.69 (3) degrees, V = 1031.1 (6) Å(3), Z = 2, D(x) = 1.406 Mg m(-3) and µ(Mo Kalpha) = 0.17 mm(-1). The final R (wR) is 0.063 (0.057) {w = 1/[sigma(2)(F) + 0.0004F(2)]} for 3619 unique reflections measured at 295 K. The aryl phosphinate group bonded to the central phenyl ring comes close to one of the two glycidyl ether groups, the epoxide ring of which is ordered. The epoxide ring far from the aryl phosphinate group is disordered. The NMR chemical shifts of the protons of the glycidyl ether group close to the aryl phosphinate group are reduced by the 'ring-current effect'.

Journal Article↗
PubMed1999

Structure of an insulin dimer in an orthorhombic crystal: the structure analysis of a human insulin mutant (B9 Ser-->Glu).

The structure of human insulin mutant B9 (Ser-->Glu) was determined by an X-ray crystallographic method at 2.5 A resolution with an R factor of 0.165 under non-crystallographic restraints. The crystals were grown at low pH (<3.8) and belong to the orthorhombic P2(1)2(1)2(1) space group with unit-cell dimensions a = 44.54, b = 46.40, c = 51.85 A and one dimer per asymmetric unit without further aggregation. The structure in this crystal form can be regarded as a model for a discrete insulin dimer and displays the following features compared with the structure of 2Zn insulin. (i) The overall dimer is expanded and more symmetric. The two A chains are about 2 A more distant from each other, while the two B chains are about 0.8 A further apart. Both monomers are more similar to molecule 1 than molecule 2 of the 2Zn insulin dimer. (ii) The dimer structure is stabilized by protonation and neutralization of the carboxyl groups at lower pH and, in addition, by formation of a hydrogen-bond network among the side chains of residues GluB9, HisB13 and HisB10 on the dimer-forming surface of both monomers, resulting from a structural rearrangement. (iii) The B-chain amino-terminal segment is in an open state (O state), i.e. a state different from the well known R and T states found in the insulin hexamer. In the O state, the B-chain N-terminal segment is in an extended conformation and is detached from the rest of the molecule. This conformational state has also been observed in the monomeric crystal structure of despentapeptide (B26-B30) and desheptapeptide (B24-B30) insulin, as well as in the solution structure of an engineered insulin monomer. It suggests that the O state may be the characteristic conformation of insulin in lower aggregation forms and may be relevant to the formation of insulin fibrils. In addition, based on the crystallization process, the smallest possible building blocks of insulin crystal are also discussed.

Amino Acid Substitution↗
PubMed2001

Sequence-structure analysis of FAD-containing proteins.

We have analyzed structure-sequence relationships in 32 families of flavin adenine dinucleotide (FAD)-binding proteins, to prepare for genomic-scale analyses of this family. Four different FAD-family folds were identified, each containing at least two or more protein families. Three of these families, exemplified by glutathione reductase (GR), ferredoxin reductase (FR), and p-cresol methylhydroxylase (PCMH) were previously defined, and a family represented by pyruvate oxidase (PO) is newly defined. For each of the families, several conserved sequence motifs have been characterized. Several newly recognized sequence motifs are reported here for the PO, GR, and PCMH families. Each FAD fold can be uniquely identified by the presence of distinctive conserved sequence motifs. We also analyzed cofactor properties, some of which are conserved within a family fold while others display variability. Among the conserved properties is cofactor directionality: in some FAD-structural families, the adenine ring of the FAD points toward the FAD-binding domain, whereas in others the isoalloxazine ring points toward this domain. In contrast, the FAD conformation and orientation are conserved in some families while in others it displays some variability. Nevertheless, there are clear correlations among the FAD-family fold, the shape of the pocket, and the FAD conformation. Our general findings are as follows: (a) no single protein 'pharmacophore' exists for binding FAD; (b) in every FAD-binding family, the pyrophosphate moiety binds to the most strongly conserved sequence motif, suggesting that pyrophosphate binding is a significant component of molecular recognition; and (c) sequence motifs can identify proteins that bind phosphate-containing ligands.

Amino Acid Motifs↗
PubMed2000

Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation.

A major structural element of bacterial endospores is a peptidoglycan (PG) wall. This wall is produced between the two opposed membranes surrounding the developing forespore and is composed of two layers. The inner layer is the germ cell wall, which appears to have a structure similar to that of the vegetative cell wall and which serves as the initial cell wall following spore germination. The outer layer, the cortex, has a modified structure, is required for maintenance of spore dehydration, and is degraded during spore germination. Theories suggest that the spore PG may also play a mechanical role in the attainment of spore dehydration. Inherent in one of these models is the production of a gradient of cross-linking across the span of the spore PG. We report analyses of the structure of PG found within immature, developing Bacillus subtilis forespores. The germ cell wall PG is synthesized first, followed by the cortex PG. The germ cell wall is relatively highly cross-linked. The degree of PG cross-linking drops rapidly during synthesis of the first layers of cortex PG and then increases two- to eightfold across the span of the outer 70% of the cortex. Analyses of forespore PG synthesis in mutant strains reveal that some strains that lack this gradient of cross-linking are able to achieve normal spore core dehydration. We conclude that spore PG with cross-linking within a broad range is able to maintain, and possibly to participate in, spore core dehydration. Our data indicate that the degree of spore PG cross-linking may have a more direct impact on the rate of spore germination and outgrowth.

Bacillus subtilis↗
PubMed1985

Human T-cell lymphotropic virus type III: immunologic characterization and primary structure analysis of the major internal protein, p24.

The major internal structural protein of human T-cell lymphotropic virus type III (HTLV-III), a virus etiologically implicated in acquired immunodeficiency syndrome (AIDS), was purified to homogeneity. This 24,000-molecular-weight protein (p24) was shown to lack immunologic cross-reacting antigenic determinants shared by other known retroviruses, including HTLV-I and HTLV-II, with the exception of equine infectious anemia virus (EIAV). A broadly reactive competition immunoassay was developed in which antiserum to EIAV was used to precipitate 125I-labeled HTLV-III p24. Although the major structural proteins of HTLV-III and EIAV competed in this assay, other type B, C, and D retroviral proteins lacked detectable reactivity. Thus, HTLV-III is more related to EIAV than to any other retroviruses. That the HTLV-III isolate is very distinct from HTLV-I and HTLV-II was further confirmed by the amino acid compositions of the major internal antigens of all three isolates. Moreover, comparison of the amino-terminal amino acid sequence of HTLV-III p24 with analogous sequences for HTLV-I and HTLV-II p24 showed that these proteins do not share significant sequence homology. In an attempt to evaluate immune response in individuals exposed to HTLV-III, sera from AIDS and lymphadenopathy syndrome patients as well as from clinically normal blood donor controls were tested for antibodies to HTLV-III p24. The results showed that sera from 93% of lymphadenopathy syndrome patients and 73% of AIDS patients exhibited high-titered antibodies to HTLV-III p24. In contrast, none of the normal control sera showed detectable reactivity to HTLV-III p24.

Acquired Immunodeficiency Syndrome↗
PubMed1984

Human alpha 1-proteinase inhibitor. Crystal structure analysis of two crystal modifications, molecular model and preliminary analysis of the implications for function.

Two closely related crystal structures of alpha 1-proteinase inhibitor modified at the reactive site peptide bond Met358--Ser359 have been analysed. The crystal structure has been obtained from diffraction data at 3 A resolution, with phases originally from isomorphous replacement. The electron density map was substantially improved by cyclic averaging of the electron densities of the two crystal forms and allowed the chain to be traced in terms of the known chemical amino acid sequence. Energy restrained crystallographic refinement was initiated and resulted in conventional R-values of 0.251 for the tetragonal crystal form (6 to 3 A resolution) and 0.247 for the hexagonal crystal form (6 to 3.2 A resolution). The polypeptide chain is almost completely arranged in well-defined secondary structural elements: three beta-sheets and eight alpha-helices. The helices are preferentially formed by the first 150 residues. They are in proximity underneath sheet A. The chain ends Met358 and Ser359 of the nicked species are arranged in strands on opposite ends of the molecule indicating a major structural rearrangement upon modification of the intact inhibitor. It is suggested that the Met358 strand is in a different conformation removed from sheet A and approaches Ser359 in the intact inhibitor species. Glu342, which is exchanged by a lysine in the Z-variant is in a strategic position for such a rearrangement. The three carbohydrate chains of alpha 1-proteinase inhibitor have partly defined electron density close to their attachment sites at asparagine residues. The anti-thrombin and ovalbumin amino acid sequences can be accommodated in the alpha 1 inhibitor molecular structure. The intron-exon junctions of the ovalbumin and the alpha 1-proteinase inhibitor gene are all in surface loops of the mature protein.

Amino Acid Sequence↗
PubMed1993

Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein.

The purine repressor protein, PurR, is a member of the lac repressor, LacI, family of Escherichia coli DNA-binding proteins that bind DNA via a highly conserved N-terminal helix-turn-helix motif. Additionally, the members of this family display strong sequence homologies between their larger C-terminal effector binding/oligomerization domains. Analysis of the PurR primary and secondary structures reveals that its C-terminal corepressor binding domain is highly homologous to another group of E. coli-binding proteins, the periplasmic binding proteins, particularly to the ribose-binding protein (RBP). The high resolution x-ray structure of RBP allows this protein to serve as a template with which to model the predicted secondary structure of the corepressor binding domain of PurR. Similarly, the N-terminal DNA binding domain of PurR can be modeled using the NMR-determined structure of the corresponding region (residues 1-56) from LacI as a template. Combining the two, results in a description of the likely secondary structure topology of PurR and implicates residues important for corepressor binding and dimerization. CD spectroscopic studies on PurR, its corepressor binding domain and RBP result in secondary structure estimates nearly identical with those obtained by sequence analyses, thereby providing further corroborating physical evidence for this topological assignment.

Amino Acid Sequence↗