The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.     

Cloning and expression of the gene for a fibronectin-binding protein from Staphylococcus aureus.

The gene encoding the fibronectin-binding protein (FNBP) from Staphylococcus aureus strain 8325-4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5-kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin-Sepharose followed by ion-exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I-labelled 165-kd polypeptide, and unlabeled 165- and 87-kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp-gene encoding the fibronectin-binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG-binding domains of protein A followed by a fibronectin-binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.     

Expression of the gene encoding protein A in Staphylococcus aureus and coagulase-negative staphylococci

Two shuttle vectors containing the gene for protein A (spa) from Staphylococcus aureus have been constructed to study expression of the gene in various strains of S. aureus and in the coagulase-negative species Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus xylosus. One plasmid, pSPA15, contains the complete structural gene for protein A, which binds to the cell wall in various Staphylococcus species. The other plasmid, pSPA16, codes for a truncated protein A lacking the C-terminal part called region X. The latter is exclusively extracellular in all Staphylococcus species tested, which confirms the importance of region X for cell wall binding. The expression of the plasmid-coded protein A in various strains of S. aureus is strongly correlated to the expression of the chromosomal spa gene. The coagulase-negative species expressing plasmid-encoded protein A produce 12 to 30% of the amount coded by the chromosomal spa gene in S. aureus strains Cowan I and A676.     

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and β-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.     

Three-dimensional model for stellacyanin, a "blue" copper-protein.

A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model.     

Background levels of hydrogen cyanide in human breath measured by infrared cavity ring down spectroscopy

Hydrogen cyanide (HCN) in breath has been suggested as a diagnostic tool for cyanide poisoning and for cyanide-producing bacterial infections. To distinguish elevated levels of breath HCN, baseline data are needed. Background levels of HCN were measured in mixed exhaled air from 40 healthy subjects (26 men, 14 women, age 21–61 years; detection limit: 1.5?ppb; median: 4.4?ppb; range <1.5–14?ppb) by near-infrared cavity ring down spectroscopy (CRDS). No correlation was observed with smoking habits, recent meals or age. However, female subjects had slightly higher breath levels of HCN than male subjects. CRDS has not previously been used for this purpose.     

Characterization of Novel CSF Tau and ptau Biomarkers for Alzheimer’s Disease

Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.     

Mutation in a flexible linker modulates binding affinity for modular complexes

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which β-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4–ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.     

Crystal structure of the topoisomerase II poison 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide bound to the DNA hexanucleotide d(CGTACG)2.

The structure of the complex formed between d(CGTACG)(2) and the antitumor agent 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide has been solved to a resolution of 1.6 A using X-ray crystallography. The complex crystallized in space group P6(4) with unit cell dimensions a = b = 30.2 A and c = 39.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The asymmetric unit contains a single strand of DNA, 1. 5 drug molecules, and 29 water molecules. The final structure has an overall R factor of 19.3%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and the protonated dimethylamino group partially occupies positions close to ( approximately 3.0 A) the N7 and O6 atoms of guanine G2. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Sugar rings adopt the C2'-endo conformation except for cytosine C1 which moves to C3'-endo, thereby preventing steric collision between its C2' methylene group and the intercalated acridine ring. The intercalation cavity is opened by rotations of the main chain torsion angles alpha and gamma at guanines G2 and G6. Intercalation perturbs helix winding throughout the hexanucleotide compared to B-DNA, steps 1 and 2 being unwound by 8 degrees and 12 degrees, respectively, whereas the central TpA step is overwound by 17 degrees. An additional drug molecule, lying with the 2-fold axis in the plane of the acridine ring, is located at the end of each DNA helix, linking it to the next duplex to form a continuously stacked structure. The protonated N,N-dimethylamino group of this "end-stacked" drug hydrogen bonds to the N7 atom of guanine G6. In both drug molecules, the 4-carboxamide group is internally hydrogen bonded to the protonated N-10 atom of the acridine ring. The structure of the intercalated complex enables a rationalization of the known structure-activity relationships for inhibition of topoisomerase II activity, cytotoxicity, and DNA-binding kinetics for 9-aminoacridine-4-carboxamides.