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Protein and Peptide Letters, Vol. 5, No. 4, 1998

Contents

Isolation and Characterization of the rpoA Gene of Mycobacterium Tuberculosis. Pp. 185-192.
Judith M. Healy, Andrea Lesoon, Jana Bodorova, Kelvin Lam and C. Richard Wobbe
[Abstract]

Chemical Synthesis of Chitin Binding Domain of Potato WIN2 Protein. Pp. 193-198.
Michiro Muraki, Hisayuki Morii and Kazuaki Harata
[Abstract]

Structural Analysis of EFB, A Fibrinogen Binding Protein of Staphylococcus aureus. Pp. 199-206.
David Wade, Marco Palma, Jan-Ingmar Flock, Kurt D. Berndt, Jerzy Silberring and Stan G. Galaktionov
[Abstract]

N-Glycosylation of the Fourth Repeat Unit of Human t Protein Abolishes Binding to the C-Terminal Acidic t-Binding Segment of b-Tubulin. Pp. 207-214.
Laszlo Otvos, Jr., Anne Marie Pease, John D. Wade and RaIf Hoffmann
[Abstract]

Immobilised and Non-Immobilised Ligand Interaction Studies of Monoclonal Antibody BC-1 and ED-B Containing Fibronectin Fragment. Pp. 215-220.
Rohanah Hussain, Giuliano Siligardi, Andrew J.T. George and Rakesh Verma
[Abstract]

Synthesis, Biological Activity and Molecular Modelling of the 53-54 Ketomethylene Analogue of HEL(52-61). Pp. 221-230.
Laurent Ettouati, Joseph Richard Casimir, Isabelle Raynaud, Marie-Claude Trescol-Biémont, Pierre-Alain Carrupt, Denis Gerlier, Chantal Rabourdin-Combe, Bernard Testa and Joelle Paris
[Abstract]

Major Structural Reorganisation Most Likely Accompanies the Transient Formation of a Physiological Electron Transfer Complex. Pp. 231-236.
Kamaldeep K. Chohan, Nigel S. Scrutton and Michael J. Sutcliffe
[Abstract]

Crystallisation Report

Expression, Purification and Preliminary Crystallographic Studies of Human Hexokinase I. Pp. 237-242.
E. Sabini, C. Rosano, C. Capasso, M. Rizzi, M. Bolognesi, M. Bianchi, G. Serafini, E. Bartolucci, and M. Magnani
[Abstract]

Abstracts

[Back to top] Isolation and Characterization of the rpoA Gene of Mycobacterium Tuberculosis. Judith M. Healy, Andrea Lesoon, Jana Bodorova, Kelvin Lam and C. Richard Wobbe.
The M. tuberculosis rpoA gene, encoding the a subunit of RNA polymerase, was isolated by a polymerase chain reaction-based strategy using primers directed against regions of a that are highly conserved among Gram-positive and Gram-negative bacteria. The amplified DNA was utilized as a hybridization probe to recover the entire rpoA gene from a cosmid library of genomic DNA from virulent M. tuberculosis strain H37Rv. Nucleotide sequencing indicated that the 1044 bp M. tuberculosis rpoA open reading frame (ORF) encodes a protein of 347 amino acids which shows significant structural similarity to the a subunits of diverse bacterial species. Recombinant M. tuberculosis a subunit was overproduced in soluble form in E. coli as a hexahistidine-tagged fusion protein and purified to homogeneity by affinity chromatography.

[Back to top] Chemical Synthesis of Chitin Binding Domain of Potato WIN2 Protein. Michiro Muraki, Hisayuki Morii and Kazuaki Harata.
The N-terminal domain of potato WIN2 protein, a 43-residue polypeptide, was synthesized by Fmoc solid phase methodology. The refolded sample in the presence of a glutathione oxidoreduction buffer was identified as the disulfides-bridged molecule accompanied with the formation of a pyroglutamate residue by MALDI-TOF mass spectrometry. The purified sample showed a strong binding activity toward solid chitin and the binding behavior was very similar to the lectin UDA.

[Back to top] Structural Analysis of EFB, A Fibrinogen Binding Protein of Staphylococcus aureus. David Wade, Marco Palma, Jan-Ingmar Flock, Kurt D. Berndt, Jerzy Silberring and Stan G. Galaktionov.
Staphylococcus aureus produces and secretes a small, basic protein, designated Efb, that binds to fibrinogen and seems to be required for virulence of the organism. A 3D model of Efb was developed, and it predicts that the C-terminal half of the protein contains substantial a-helical content. CD analysis of Efb yielded a value of 40-41% a-helix. Calcium and zinc both influence the interactions between Efb and fibrinogen, and an analysis of the amino acid sequence of Efb revealed the presence of consensus sequences for the binding of both metals.

[Back to top] N-Glycosylation of the Fourth Repeat Unit of Human t Protein Abolishes Binding to the C-Terminal Acidic t-Binding Segment of b-Tubulin. Laszlo Otvos, Jr., Anne Marie Pease, John D. Wade and RaIf Hoffmann.
Tubulin binds to the 18-amino acid repeat units of normal t protein through several sites. Fluorescence polarization showed t repeats 1, 3, and 4 bound to an acidic dodecapeptide fragment of tubulin in mM concentrations. From the proposed abnormal modifications of t in Alzheimer's disease, only addition of an N-acetyl-glucosamine moiety to the potential N-glycosylation site Asn359 abolished the t-tubulin interaction indicating the seminal role of overutilization of this site during the development of the paired helical filaments.

[Back to top] Immobilised and Non-Immobilised Ligand Interaction Studies of Monoclonal Antibody BC-1 and ED-B Containing Fibronectin Fragment. Rohanah Hussain, Giuliano Siligardi, Andrew J.T. George and Rakesh Verma.
The interaction of antibody BC-1 with ED-B containing fibronectin fragment was successfully monitored by a non-immobilised ligand interaction assay by CD spectroscopy (NILIA-CD) and by an immobilised ligand assay using IAsys resonant mirror biosensor. NILIA-CD not only could be used to determine equilibrium constant of the interaction but also indicated changes in both backbone and aromatic side-chain regions upon binding. The biosensor, however, provides the association/dissociation kinetics of the interacting systems. Both techniques are complementary to each other in enabling maximum extraction of ligand/host interaction information.

[Back to top] Synthesis, Biological Activity and Molecular Modelling of the 53-54 Ketomethylene Analogue of HEL(52-61). Laurent Ettouati, Joseph Richard Casimir, Isabelle Raynaud, Marie-Claude Trescol-Biémont, Pierre-Alain Carrupt, Denis Gerlier, Chantal Rabourdin-Combe, Bernard Testa and Joelle Paris.
The synthesis of the 53-54 ketomethylene isostere of HEL(52-61), an antigenic decapeptide, was realised by convergent Fmoc solid-phase peptide synthesis. In addition to the target peptide, an unexpected hydroxysuccinyl by-product was recovered. The two ketomethylene epimers did not exhibit any stimulating or competitive activity. Molecular modelling studies of the analogues-MHC-II I-Ak complex suggested a departure from the canonical polyproline II conformation which could account for the absence of binding to I-Ak molecule.

[Back to top] Major Structural Reorganisation Most Likely Accompanies the Transient Formation of a Physiological Electron Transfer Complex. Kamaldeep K. Chohan, Nigel S. Scrutton and Michael J. Sutcliffe.
Modelling studies coupled with experimental observations show that the transfer of electrons from trimethylarnine dehydrogenase to its electron transferring flavoprotein (ETF) requires a large quaternary change in ETF. These studies describe for the first time how complex and substantial changes in quaternary structure could control interprotein electron transfer reactions, and direct electrons onto specific and intended redox partners.

[Back to top] Expression, Purification and Preliminary Crystallographic Studies of Human Hexokinase I. E. Sabini, C. Rosano, C. Capasso, M. Rizzi, M. Bolognesi, M. Bianchi, G. Serafini, E. Bartolucci, and M. Magnani.
Human hexokinase type 1, one of the four isozymes consisting of a single polypeptide chain of about 100 kDa, has been cloned in the pET expression plasmid in a truncated form lacking a segment of II apolar amino acids at the N-terminus. The protein has been overexpressed in E.coli and purified to homogeneity. Truncated hexokinase I has been crystallized in the presence of glucose 6-phosphate and of the ATP analogue AMP-PNP. The crystals belong to the monoclinic space group P21, with unit cell constants: a = 84.2 Å, b = 177.7 Å, c = 88.2 Å, b = 90.8° and diffract to 3.0 Å resolution.