Membrane activity of the peptide antibiotic clavanin and the importance of its glycine residues.

The peptide antibiotic clavanin A (VFQFLGKIIHHVGNFVHGFSHVF-NH(2)) is rich in histidine and glycine residues. In this study the antimicrobial activity and membrane activity of wild-type clavanin A and seven Gly --> Ala mutants thereof were investigated. Clavanin A effectively killed the test microorganism Micrococcus flavus and permeabilized its cytoplasmic membrane in the micromolar concentration range, suggesting that the membrane is the target for this molecule. Consistent with this suggestion, it was observed that clavanin A efficiently inserted into different phospholipid monolayers mainly via hydrophobic interactions. Bilayer permeabilization was observed for both low and high molecular mass fluorophores enclosed in unilamellar vesicles and occurred at the same concentration as the antimicrobial activity. It is therefore suggested that the loss of barrier function does not involve specific receptors in the target membrane. Circular dichroism spectroscopy indicated that under membrane mimicking conditions a random coil --> helical transition was induced for all clavanin derivatives tested. Observed differences in peptide-membrane interaction and biological activity between the various clavanin derivatives demonstrated the functional importance of Gly at the positions 6 and 13. These two glycines may act as flexible hinges that facilitate the hydrophobic N-terminal end of clavanin to deeply insert into the bilayer. On the contrary, no such role is evident for Gly 18, as its substitution by Ala actually stimulated membrane interaction and biological activity. This study suggests that the combined hydrophobicity, overall state of charge, and conformational flexibility of the peptide determine the (membrane) activity of clavanin A and its Gly --> Ala mutants.

Interaction of beta-lactoglobulin with small hydrophobic ligands as monitored by fluorometry and equilibrium dialysis: nonlinear quenching effects related to protein--protein association.

Although a thorough characterization of binding parameters is essential for application of beta-lactoglobulin as a carrier for a variety of small hydrophobic ligands, the binding parameters derived in various studies using various techniques are inconsistent. The bindings of several small ligands as detected by fluorometry and equilibrium dialysis were compared. Fluorescence spectroscopy showed that beta-ionone, retinol, and fatty acid lactones all bound in the vicinity of a tryptophan residue. Retinol and fatty acid lactone competed for the same binding site. Exclusively for ligands that quench the beta-lactoglobulin fluorescence through a resonance energy transfer mechanism, fluorometry yielded a systematically higher binding affinity than equilibrium dialysis. The binding overestimation in fluorometric measurements can be explained by oligomer formation of protein, together with an underestimation of the limiting quenching level at saturating ligand concentrations due to the use of a limited set of data points.

Vernier zone residue 4 of mouse subgroup II kappa light chains is a critical determinant for antigen recognition.

BACKGROUND: During the conversion of murine monoclonal antibodies directed against the human chorionic gonadotropin (hCG) into bacterially expressed single chain fragments (scFv), we found a major reduction of binding activity upon introduction of a primer encoded mutation. OBJECTIVES: In this study we tried to determine which mutation was responsible and on what manner this mutation affected antigen binding (structural effect versus direct involvement of the residue in binding). RESULTS: No binding could be detected, when the wild type residue methionine at position 4 within the Framework region 1 of the Vkappa light chain was substituted by serine in two antibodies with a subgroup II kappa light chain. However, a similar replacement within an anti-hCG antibody with a subgroup IV kappa light chain and thereby having leucine as wild type residue, did not affect the binding characteristics. The mutant scFv's derived from both AB-s sensitive for substitution by serine never reacted with antigen in ELISA. Analysis with surface plasmon resonance revealed a residual binding only on a sensorchip with a high density coating of antigen; however, an increased dissociation, relative to that of the wild type scFv and the absence of reactivity in ELISA suggest a drastically altered affinity. CONCLUSION: A structural explanation for the changed binding characteristics can be the influence of the position 4 residue, as being a constituent of the Vernier zone, on the position of the CDR1 loop of Vkappa, which might harbour residues that directly bind to antigen, or indirectly positions other variable loops of the binding pocket. An increased sensitivity for trypsin digestion supported the hypothesis of a local conformational change in the serine mutant of the subgroup II kappa containing antibody.

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding.

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.

Molecular modelling of CCK-A receptors.

Recently, the primary structure of the cholecystokinin A-type (CCK-A) receptor has been determined. From the Kyte-Doolittle-predicted hydrophobic stretches of this sequence and the transmembrane domains of bacteriorhodopsin, a membrane-bound protein of known tertiary structure, a three-dimensional model of the membrane-embedded part of this receptor was built. Subsequently, the modelled receptor pore was searched for a binding site that matches the structural and conformational characteristics of the parent classes of the antagonists devazepide and lorglumide. In addition, the binding mode of hybrid analogues of these reference compounds was examined. The proposed antagonist, binding site includes regions in which hydrophobic, hydrogen-bonding and aromatic interactions stabilize the receptor-ligand complex.

Molecular modelling of asperlicin derived cholecystokinin A receptor antagonists.

The C3-substituted benzodiazepines derived from asperlicin, e.g. devazepide (L-364,718, MK-329), constitute the most potent class of cholecystokinin A-type (CCKA) receptor antagonists. In order to gain insight into the prerequisites for binding, we examined the conformational properties of both potent and weak representatives of this class with computer assisted molecular modelling (CAMM) techniques. The CAMM results indicate that the binding site for the C3-substituents is a planar slot on the CCKA receptor surface and, in addition, allow the proposal of a model which describes the relative binding mode of the less potent R isomers versus that of the S isomers. The latter model illustrates the unique spatial properties of the benzodiazepine moiety, which we suggest functions primarily as an invertible core which assures an optimal arrangement of attached substituents.

Hybrid cholecystokinin-A antagonists based on molecular modeling of lorglumide and L-364,718.

A series of novel nonpeptide cholecystokinin-A (CCK-A) antagonists have been synthesized. Designed on the basis of the structural homology between lorglumide and L-364,718, as investigated with molecular modeling, these compounds constitute a link between the N-acylglutamic acid and 3-amino-5-phenyl-1,4-benzodiazepin-2-one derived antagonists. The prepared compounds were tested in vitro as antagonists of the binding of [3H]-(+/-)-L-364,718 and [3H]-CCK-8(S) to rat pancreas and guinea pig brain membranes, respectively. All compounds proved to be selective for the (peripheral) CCK-A receptor, the most potent analogue, 6, having a Ki value of 90 nM. The structure-activity profile of the series of hybrid compounds relates closest to that of the N-acylglutamic acid derived antagonists.