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C L Lawson

Publications and source records attributed to C L Lawson.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi.

Outer surface protein C (OspC) is a major antigen on the surface of the Lyme disease spirochete, Borrelia burgdorferi, when it is being transmitted to humans. Crystal structures of OspC have been determined for strains HB19 and B31 to 1.8 and 2.5 A resolution, respectively. The three-dimensional structure is predominantly helical. This is in contrast to the structure of OspA, a major surface protein mainly present when spirochetes are residing in the midgut of unfed ticks, which is mostly beta-sheet. The surface of OspC that would project away from the spirochete's membrane has a region of strong negative electrostatic potential which may be involved in binding to positively charged host ligands. This feature is present only on OspCs from strains known to cause invasive human disease.

Amino Acid Sequence↗
PubMed2001

Structural conservation of neurotropism-associated VspA within the variable Borrelia Vsp-OspC lipoprotein family.

Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to avoid the immune response. One of these proteins, VspA of Borrelia turicatae, is also associated with neurotropism in infected mice. Vsp proteins are highly polymorphic in sequence, which may relate to their specific antibody reactivities and host cell interactions. To determine whether sequence variations affect protein structure, we compared B. turicatae VspA with three related proteins: VspB of B. turicatae, Vsp26 of the relapsing fever agent Borrelia hermsii, and OspC of the Lyme disease spirochete Borrelia burgdorferi. Recombinant non-lipidated proteins were purified by affinity or ion exchange chromatography. Circular dichroism spectra revealed similar, highly alpha-helical secondary structures for all four proteins. In vitro assays demonstrated protease-resistant, thermostable Vsp cores starting at a conserved serine at position 34 (Ser(34)). All proteins aggregate as dimers in solution. In situ trypsin treatment and surface protein cross-linking showed that the native lipoproteins also form protease-resistant dimers. These findings indicate that Vsp proteins have a common compact fold and that their established functions are based on localized polymorphisms. Two forms of VspA crystals suitable for structure determination by x-ray diffraction methods have been obtained.

Amino Acid Sequence↗
PubMed2000

Structural identification of a key protective B-cell epitope in Lyme disease antigen OspA.

Outer surface protein A (OspA) is a major lipoprotein of the Borrelia burgdorferi spirochete, the causative agent of Lyme disease. Vaccination with OspA generates an immune response that can prevent bacterial transmission to a mammalian host during the attachment of an infected tick. However, the protective capacity of immune sera cannot be predicted by measuring total anti-OspA antibody. The murine monoclonal antibody LA-2 defines an important protective B-cell epitope of OspA against which protective sera have strong levels of reactivity. We have now mapped the LA-2 epitope of OspA using both NMR chemical-shift perturbation measurements in solution and X-ray crystal structure determination. LA-2 recognizes the three surface-exposed loops of the C-terminal domain of OspA that are on the tip of the elongated molecule most distant from the lipid-modified N terminus. The structure suggests that the natural variation at OspA sequence position 208 in the first loop is a major limiting factor for antibody cross-reactivity between different Lyme disease-causing Borrelia strains. The unusual Fab-dominated lattice of the crystal also permits a rare view of antigen flexibility within an antigen:antibody complex. These results provide a rationale for improvements in OspA-based vaccines and suggest possible designs for more direct tests of antibody protective levels in vaccinated individuals.

Amino Acid Sequence↗
PubMed2000

High-diffraction-efficiency pseudorandom encoding.

Pseudorandom encoding (PRE) is a statistics-based procedure in which a pure-phase spatial light modulator (SLM) can yield, on the average, the prescribed diffraction pattern specified by the user. We seek to combine PRE with the optimization of an aperture-based target function. The target function is a fully complex input transmittance, unrealizable by a phase-only SLM, that generates a prescribed light intensity. The optimization is done to increase the diffraction efficiency of the overall process. We compare three optimization methods-Monte Carlo simulation, a genetic algorithm, and a gradient search-for maximizing the diffraction efficiency of a spot-array generator. Calculated solutions are then encoded by PRE, and the resulting diffraction patterns are computer simulated. Details on the complexity of each procedure are furnished, as well as comparisons on the quality, such as uniformity of the output spot array.

Computer Simulation↗
PubMed1999

Model for lentivirus capsid core assembly based on crystal dimers of EIAV p26.

Two crystal forms of recombinant p26 capsid protein (CA) from the equine infectious anemia virus (EIAV) have in common an antiparallel four-helix bundle dimer interface between N-terminal domains (NTDs). The dimer interface provides a lenient scaffold to accommodate the wide sequence variation in these helices within lentivirus CA. Pairs of dimers weakly associate to form exact or approximate D2 symmetry tetramers. In one of the two crystal forms, the tetramers are linked via dimerization of C-terminal domains (CTDs). We propose that the observed NTD and CTD homodimer interactions are involved in the assembly of the lentivirus capsid. The NTD homodimer shape readily suggests a model for the mature capsid core, based on hexagonal packing with dimensions and surface topology resembling described EIAV capsid cores. Combining available data for human immunodeficiency virus and EIAV CA, we also propose an assembly pathway for maturation of the lentivirus capsid core following proteolytic cleavage of the gag polyprotein precursor.

Amino Acid Sequence↗
PubMed1997

Crystal structure of Lyme disease antigen outer surface protein A complexed with an Fab.

OspA (outer surface protein A) is an abundant immunogenic lipoprotein of the Lyme disease spirochete Borrelia burgdorferi. The crystal structure of a soluble recombinant form of OspA was solved in a complex with the Fab fragment of mouse monoclonal antibody 184.1 and refined to a resolution of 1.9 A. OspA has a repetitive antiparallel beta topology with an unusual nonglobular region of "freestanding" sheet connecting globular N- and C-terminal domains. Arrays of residues with alternating charges are a predominant feature of the folding pattern in the nonglobular region. The 184.1 epitope overlaps with a well conserved surface in the N-terminal domain, and a hydrophobic cavity buried in a positively charged cleft in the C-terminal domain is a potential binding site for an unknown ligand. An exposed variable region on the C-terminal domain of OspA is predicted to be an important factor in the worldwide effectiveness of OspA-based vaccines.

Amino Acid Sequence↗
PubMed1995

Site-directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 affect activity and product specificity.

Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae.

Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form.

The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.

Amino Acid Sequence↗
PubMed1993

Cocrystals of Escherichia coli trp repressor bound to an alternative operator DNA sequence.

Cocrystals of a 2:1 complex of trp repressor dimers with a DNA duplex containing a single, central operator half-site sequence are described. Crystals with different morphologies grew under diverse crystallization conditions within days to weeks by hanging-drop vapor-diffusion. Twinned rods split along their longitudinal cleft produce single crystals with space group C2 and unit cell dimensions a = 112.34 A, b = 90.16 A, c = 58.65 A and beta = 113.92 degrees. The crystals diffract to 2.4 A and are thus suitable for structural analysis by X-ray diffraction. Several heavy-atom derivative cocrystals have been obtained with iodouridine-substituted DNAs.

Bacterial Proteins↗
PubMed1993

Tandem binding in crystals of a trp repressor/operator half-site complex.

The crystal structure of trp repressor tandemly bound in a 2:1 complex to a 16-base-pair palindromic DNA containing a central trp operator half-site has been determined and refined to 2.4 A resolution. Despite dramatically different DNA sequence contexts and crystallization conditions, the protein/DNA interface is essentially identical to that seen in the original trp repressor/operator complex structure. Water-mediated sequence recognition by trp repressor is likely to be related to the unusual end-on approach of the recognition helix (E), which allows sharing of the major groove by tandem dimers. The tandem complex model accounts for the mutational sensitivity of all trp operator base pairs. The structure also provides the first detailed view of the tandem interaction, revealing a key role for the amino-terminal arms.

Bacterial Proteins↗
PubMed1990

Maltodextrin-dependent crystallization of cyclomaltodextrin glucanotransferase from Bacillus circulans.

Crystals of cyclomaltodextrin glucanotransferase from Bacillus circulans (EC 2.4.1.19) suitable for high-resolution X-ray analysis were obtained by vapor diffusion against 60% (v/v) 2-methyl 2,4-pentanediol buffered with 100 mM-sodium Hepes, pH 7.55. The crystals have P2(1)2(1)2(1) space group symmetry, with a = 120.4 A, b = 110.9 A and c = 66.4 A, and contain one molecule of 68,000 in the asymmetric unit. Growth of single enzyme crystals was found to require the presence of either alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or maltose in high molar excess, a requirement that could not be fulfilled by glucose, the basic building block of these compounds. Although the exact role of cyclic and linear maltodextrins in enzyme crystallization is not yet known, we have preliminary evidence that these compounds are degraded by the enzyme in the crystallization droplet.

Bacillus↗
PubMed1989

Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase.

Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 A. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.

Alcohol Oxidoreductases↗
PubMed1988

Crystal structure of trp repressor/operator complex at atomic resolution.

The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface. Water-mediated polar contacts to the bases also appear to contribute part of the specificity.

Crystallography↗
PubMed1988

The structure of trp pseudorepressor at 1.65A shows why indole propionate acts as a trp 'inducer'.

The trp repressor is a small dimeric regulatory protein which controls the expression of three operons in Escherichia coli. The inactive aporepressor protein must bind two molecules of L-tryptophan to form the active repressor. If desamino analogues of L-tryptophan such as indole propionate (IPA) are substituted for L-tryptophan, an inactive pseudorepressor is formed. Because the desamino analogues thus cause derepression of operons under control of the trp repressor, they appear to be 'inducers'. We have determined the crystal structure of the pseudorepressor and refined it to 1.65 A. The molecular structure was compared to that of the nearly isomorphous orthorhombic form of the repressor. Surprisingly, the indole ring of IPA is in the same position as the indole ring of L-tryptophan in the repressor, but is 'flipped over'. As a result, the carboxyl group of IPA is oriented toward the DNA-binding surface of the protein and is in a position where it sterically and electrostatically repels the phosphate backbone of both operator and non-operator DNA. This explains why IPA acts as an apparent trp inducer.

Apoproteins↗
PubMed1988

Flexibility of the DNA-binding domains of trp repressor.

An orthorhombic crystal form of trp repressor (aporepressor plus L-tryptophan ligand) was solved by molecular replacement, refined to 1.65 A resolution, and compared to the structure of the repressor in trigonal crystals. Even though these two crystal forms of repressor were grown under identical conditions, the refined structures have distinctly different conformations of the DNA-binding domains. Unlike the repressor/aporepressor structural transition, the conformational shift is not caused by the binding or loss of the L-tryptophan ligand. We conclude that while L-tryptophan binding is essential for forming a specific complex with trp operator DNA, the corepressor ligand does not lock the repressor into a single conformation that is complementary to the operator. This flexibility may be required by the various binding modes proposed for trp repressor in its search for and adherence to its three different operator sites.

Apoproteins↗
PubMedDate not supplied

Crystallization and preliminary X-ray analysis of Borrelia burgdorferi outer surface protein A (OspA) complexed with a murine monoclonal antibody Fab fragment.

The Borrelia burgdorferi outer surface lipoprotein OspA is a current focus for vaccine development to prevent Lyme disease infection. A soluble, recombinant form of the protein lacking the amino-terminal lipid membrane anchor was cocrystallized with the Fab fragment of an agglutinating mouse monoclonal antibody. The crystals belong to space group P2(1)2(1)2(1), with a = 90.0 A, b = 91.9 A, and c = 102.9 A and they were found to diffract to a maximum resolution of 2.8 A using synchrotron radiation.

Animals↗