A conformationally isoformic thermophilic protein with high kinetic unfolding barriers.

The basis for the stability of thermophilic proteins is of fundamental interest for extremophile biology. We investigated the folding and unfolding processes of the homotetrameric Thermoanaerobacter brockii alcohol dehydrogenase (TBADH). TBADH subunits were 4.8 kcal/mol less stable towards guanidinium chloride (GdmCl) unfolding compared to urea, indicating ionic modulation of TBADH stability. Strongly denaturing conditions promoted mono-exponential unfolding kinetics with linear dependence on denaturant concentration. Here TBADH unfolded >40-fold slower when extrapolated from urea as compared to GdmCl unfolding. A marked unfolding hysteresis was shown when comparing refolding and unfolding in urea. An unusual biphasic unfolding trajectory with an exceptionally slow phase at intermediate concentrations of GdmCl and urea was also observed. We advocate that TBADH forms two distinctly different tetrameric isoforms, and likely an ensemble of native states. This unusual supramolecular folding behavior has been shown responsible for formation of amyloidotic yeast prion strains and can have functional importance for TBADH.

Molecularly imprinted polymers--tyrosinase mimics.

Synthetic polymers mimicking the enzyme tyrosinase have been prepared by the molecular imprinting of a complex between Cu (II) and catechol and ethyl ester of urocanic acid in an ethylene glycol dimethacrylate copolymer. Optimised polymer systems demonstrated catalysis, Michaelis-Menten kinetics and competitive inhibition similar to those of mushroom tyrosinase. The polymers benefited from superior chemical and mechanical stability in comparison with natural enzyme.

Filamentous bacteriophage stability in non-aqueous media.

BACKGROUND: Filamentous bacteriophage are used as general cloning vectors as well as phage display vectors in order to study ligand-receptor interactions. Exposure to biphasic chloroform-water interface leads to specific contraction of phage, to non-infective I- or S-forms. RESULTS: Upon exposure, phage were inactivated (non-infective) at methanol, ethanol and 1-propanol concentrations inversely dependent upon alcohol hydrophobicity. Infectivity loss of phage at certain concentrations of 1-propanol or ethanol coincided with changes in the spectral properties of the f1 virion in ultraviolet fluorescence and circular dichroism studies. CONCLUSIONS: The alcohols inactivate filamentous phage by a general mechanism--solvation of coat protein--thereby disrupting the capsid in a manner quite different from the previously reported I- and S-forms. The infectivity retention of phagemid pG8H6 in 99% acetonitrile and the relatively high general solvent resistance of the phage strains studied here open up the possibility of employing phage display in non-aqueous media.

Molecular imprinting of surfaces.

Recent years have seen the development of a number of novel strategies for improving the performance of molecularly imprinted polymers and for adapting them to new application areas. The imprinting of surfaces has received significant attention over the past few years, due to the possibilities available in terms of the imprinting of macromolecular structures and for avoiding problems with mass transfer. Furthermore, some surface imprinting protocols offer unique possibilities for a number of application areas. In this review we present an analysis of the approaches thusfar employed for the imprinting of surfaces and discuss the consequences of these strategies for application development.

Towards the rational design of molecularly imprinted polymers.

Efforts to elucidate the mechanisms underlying the formation of binding sites in molecularly imprinted polymers (MIPs) and of MIP-ligand binding events are presented in the context of a thermodynamic treatment of MIP recognition phenomena.

Novel chiral recognition elements for molecularly imprinted polymer preparation.

The use of a novel chiral functional monomer system in molecular imprinting protocols is described. The monomer, dibenzyl (2R,3R)-O-monoacryloyl tartrate, possesses a hydroxyl moiety which can be used to direct template-functional monomer interactions during molecular imprinting polymerization. This system simultaneously positions benzyl ester-protected carboxyl groups in close proximity to the template, which upon deprotection yield recognition sites with stronger ligand-binding capacities. Furthermore, the inherent chirality of the monomer engenders the polymer with an inbuilt preference for a given stereoisomer. Application of the system to the molecular imprinting of the cinchonidine alkaloids (+)-cinchonine and (-)-cinchonidine yielded stereoselective polymers. The effect of imprinting (+)-cinchonine produced a polymer which more than reversed the inherent chiral selectivity of the chiral monomer residues present in the matrix.

Spectroscopic studies of the molecular imprinting self-assembly process.

A method for the rapid estimation of the extent of complex formation in molecular imprinting prepolymerization mixtures is described. By the use of a UV spectroscopy titration procedure, apparent binding constants for such self-assembly processes have been obtained. This method was used for comparison of the interactions between a dipeptide template (N-acetyl-L-phenylalaninyl-L-tryptophanyl methyl ester) and the functional monomer methacrylic acid, and the monomer analogues acetic acid and trifluoroacetic acid. The importance of template-monomer association during the molecular imprinting prepolymerization phase is discussed with respect to the systems studied.

Phage viability in organic media: insights into phage stability.

The stability of the filamentous phages derived from phagemid pG8H6 has been examined in a range of solvents and solvent mixtures. The results show an enhanced capacity to infect E. coli after exposure to various organic solvent-water mixtures. The dependence of stability upon solvent hydrophobicity was demonstrated. Furthermore, conditions have been identified which should allow the application of phage display libraries based upon pG8H6 in organic media.

The rational use of hydrophobic effect-based recognition in molecularly imprinted polymers.

A novel molecularly imprinted polymer (MIP) system selective for D-phenylalanine is described where polymerization is performed in aqueous solution. The unique polymer system comprises a hydrophobic moiety-selective functional monomer, polymerizable beta-cyclodextrin, an electrostatic interacting functional monomer, 2-acryloylamido-2-methylpropane sulfonic acid (AMPSA), and the crosslinking agent N,N'-diacryloylpiperazine. Chromatographic evaluation of polymer-ligand recognition characteristics demonstrated ligand selectivity by the MIP and that optimal recognition was achieved through a balance of hydrophobic and electrostatic ligand-polymer interactions, indicating that recognition in these systems is regulated by enthalpy-entropy compensation. The imprinting effect was shown to be sufficient to reverse the inherent selectivity of cyclodextrin for L-phenylalanine.

Theophylline molecularly imprinted polymer dissociation kinetics: a novel sustained release drug dosage mechanism.

The template release kinetics of theophylline molecularly imprinted polymers has been examined with a view to determining their potential as a controlled release drug dosage form. The basis for the ligand selectivity of these polymers has been shown through the demonstration of pre-polymerization template-monomer complexation and HPLC studies of the product polymer ligand selectivities. The release kinetics shows a dependence upon template loading and pH. Small differences in release characteristics between imprinted and non-imprinted (reference) polymers have been observed.

Some recent developments in the preparation of novel recognition systems: a recognition site for the selective catalysis of an aldol condensation using molecular imprinting and specific affinity motifs for alpha-chymotrypsin using a phage display peptide library.

Molecular imprinting and phage display library technologies are rapidly being accepted as useful techniques for the generation of ligand-selective recognition motifs. The use of molecular imprinting to produce a novel type II aldolase mimic selective for the cobalt(II)-mediated aldol condensation of benzophenone and acetaldehyde is reported here. Furthermore, peptide motifs have been identified which are acting as 'affinity ligands' selective for the recognition of the enzyme alpha-chymotrypsin using phage display techniques.

NMR and molecular modelling based conformational analysis of some N-alkyl 1- and 2-benzazepinones: useful central nervous system agent design motifs.

Variable temperature 1H NMR lineshape analyses and 13C NMR spin-lattice relaxation time studies were undertaken to characterise the conformationally dynamic N-alkyl 1- and 2-benzazepinones 1 and 2. For 1, dynamic interchange between two mirror-image puckered forms of the azepine ring occurs with a barrier of 56 kJ mol-1. There is a significantly greater degree of ring flexibility in the case of 2. Molecular modelling studies were used to examine the degree of sidechain flexibility in these compounds.

Central nervous system receptor binding profiles of some 2-amino-4-phenyl quinolines: a novel class of alpha 2-adrenoceptor selective ligands.

The 2-amino-4-phenyl quinoline moiety is a structural motif common to a number of central nervous system active agents. Extensive radio-ligand receptor binding profiles for several derivatives of this common structural feature have been determined. A high base level of central nervous system receptor affinity was observed with a distinct preference for the alpha-, and in particular the alpha 2-, adrenoceptors.