Membrane channel forming polypeptides studies on alamethicin, antiamoebin and related peptides

Abstract

Membrane channel forming polypeptides possessing ionophoric activity have been widely investigated as models for transmembrane ion transport and for their effects on both artificial and natural membranes. This thesis describes investigations on alamethicin and related polypeptides, with emphasis on their effects on mitochondrial oxidative phosphorylation and on ionophoric activity in liposomal membranes. A detailed study of the solution conformation of a natural aminoisobutyric acid (Aib)–containing peptide, antiamoebin, has also been undertaken. This thesis is divided into seven chapters.

Chapter I – Introduction Chapter I provides a detailed introduction to the structural chemistry and membrane modifying activity of the channel forming peptides belonging to the alamethicin family of antibiotics. A brief background on linear gramicidins and melittin is also included.

Chapter II – Alamethicin and Oxidative Phosphorylation After a brief introduction to mitochondrial oxidative phosphorylation and classical uncouplers, this chapter describes the effects of alamethicin on oxidative phosphorylation in rat liver mitochondria over a wide range of inorganic phosphate (Pi) concentrations (2.5–100 mM). Key findings:

Increasing Pi concentration induces a transition from uncoupling to inhibition of mitochondrial respiration by alamethicin. Classical uncoupling is observed at low Pi (2.5–25 mM). Inhibitory effects predominate at higher Pi (50–100 mM).

Chapter III – Comparison with Other Peptide Ionophores The mitochondrial effects of alamethicin are compared with those of gramicidin A, melittin, and its tetraacetyl and tetrasuccinyl derivatives. Uncoupling efficiency at low peptide concentrations follows the order: gramicidin A >> alamethicin > tetraacetyl melittin > melittin > tetrasuccinyl melittin 0 Ca² flux across lipid bilayers was monitored using the increase in chlorotetracycline (CTC) fluorescence upon Ca² binding. The ability to induce Ca² transport follows the order: melittin > tetraacetyl melittin > tetrasuccinyl melittin Mitochondrial swelling assays were also performed. Additional findings:

Tetraacetyl melittin exhibits alamethicin like behaviour, showing a transition from uncoupling to inhibition at higher concentrations. Melittin shows a less pronounced inhibitory effect. Gramicidin A shows no such transition. The major site of inhibition is Complex IV (cytochrome oxidase), with moderate effects at the Complex II–III segment.

The results are interpreted in terms of Pi transport mediated by peptide channels.

Chapters IV & V – Antiamoebin: Structure and Activity Antiamoebin is a fungal antibiotic rich in Aib residues.

Chapter IV – Fractionation and Biological Activity Antiamoebin was fractionated by HPLC into four closely related components. Key observations:

All fractions exhibit ionophoric activity. Each fraction induces Ca² transport across lipid bilayers. Each fraction uncouples mitochondrial oxidative phosphorylation. Differences in membrane modifying activities are evident at low peptide concentrations.

This study provides the first evidence for the ionophoric properties of antiamoebin.

Chapter V – NMR Conformational Analysis of Antiamoebin Using 1D and 2D NMR in dimethyl sulfoxide, the conformational features of antiamoebin were elucidated. Key findings:

Ten backbone NH groups are intramolecularly hydrogen bonded. Three NH groups-Phe(1), Aib(2), and Aib(8)-are free. Inter residue NOEs correspond to values of approx. –120° ± 30° at Phe(1) and Leu(7). The stereochemical constraints imposed by Aib, isovalyl, prolyl, and 4 hydroxyprolyl residues favour a highly ordered helical structure. Two backbone conformations consistent with the data are discussed.

Chapter VI – Synthetic Aib Containing Peptides This chapter compares the membrane modifying activities of several synthetic Aib peptides, differing in chain length and sequence. Findings:

Synthetic emerimicin fragments uncouple mitochondrial oxidative phosphorylation. A minimum chain length of 7 residues is required for uncoupling activity, with efficiency increasing with chain length. For zervamicin fragments, the trend is different:

A 10 residue peptide (ZA1 10) shows greater ionophoric activity than a longer 16 residue peptide (ZA1 16).

Among alternating (Aib X) peptides, only the 10 mer (V ) shows Ca² transport.

Comparison of 10 residue peptides shows the following order of Ca² transport: V > ZA1 10 > E

Chapter VII – Summary Chapter VII summarises the major findings:

Alamethicin and related peptides display dual behaviour depending on Pi concentration. Melittin derivatives show graded ionophoric and uncoupling activity. Antiamoebin exhibits strong ionophoric behaviour and adopts a highly ordered conformation. Synthetic Aib peptides show chain length and sequence dependent activity.

These studies deepen understanding of Aib rich ionophoric peptides, their conformational properties, and their effects on biological and model membranes.