Flurorescence investigation of biological systems. the alamethicin transmembrane channel and model systems

Abstract

Fluorescence is a technique that has been extensively utilized in the study of systems of biological relevance due to the longevity of the excited states of many molecules, which allows for a variety of physical and chemical interactions to occur during the lifetime of the excited state. This thesis is concerned with the application of fluorescence techniques to elucidate the mechanisms of channel?mediated ion transport through membranes and to examine interactions in model systems such as micelles, liposomes, and serum albumins.

The thesis is presented in two parts.

SECTION I addresses the problem of ion transport through membranes. The system chosen is the alamethicin channel. Considerable work has been done in this laboratory on the structure of synthetic fragments of alamethicin and a related polypeptide antibiotic, suzukacillin. These peptide ionophores are presumed to aggregate to form transmembrane, water?filled pores stabilized by hydrophobic interactions among the monomer units. This section attempts to correlate the structural requirements for channel?forming ability of these peptides so as to arrive at a model for the channel.

The Section is preceded by an introduction to ion transport through membranes. The use of CTC as a fluorescent probe of divalent cations has been reinvestigated as part of a program to apply CTC fluorescence to monitor Ca²? fluxes across lipid membranes. Chapter II describes spectral studies on CTC. A hitherto unreported band emitting at 430 nm above pH 7.5 is reported and assigned to the BCD ring system of the molecule. The pH, solvent, and cation dependence of CTC have been studied. Energy transfer from the 430 nm band to the 530 nm band in the molecule is detected and found to be facilitated under conditions favoring 1:1 complex formation with di? and trivalent cations. Binding of cations has been studied by absorption spectroscopy and an estimate made of the size of the binding cavity.

In Chapter III a method is developed for monitoring divalent cation fluxes across liposomal membranes utilizing the increase in CTC fluorescence on binding divalent cations. Correlations with electrical conductivity measurements on planar bilayer lipid membranes (BLM) have been drawn. The initial slope of the fluorescence increase corresponds to transmembrane current while transmembrane potentials can be generated by varying the ionic composition across the membrane. The stoichiometry of the X537A cation translocating unit is found to be 2:1, with poor ionophore selectivity between Ca²?, Zn²?, and La³?. An exponential dependence of transmembrane current on transmembrane potential is obtained for alamethicin as earlier reported using BLM.

Chapter IV derives structure?activity relationships for synthetic alamethicin fragments. Divalent cation fluxes using the method described in Chapter III have been studied in small unilamellar liposomes, synaptosomes, and intact sperm cells. Channel formation has also been followed using the uncoupling of oxidative phosphorylation as a parameter. A minimum chain length of 13 residues is required for activity; negative charges on free carboxylates are inhibitory; hydrophobicity conferred either by increasing chain length or by N?protecting groups favors the formation of channels. These structural factors also favor aqueous?phase aggregation.

Aqueous?phase aggregation of N?dansylglycyl labelled fragments of alamethicin has been followed fluorimetrically in Chapter V. N?dansylglycyl peptides are still active; their activity parallels those of the parent peptides in both assay systems described in Chapter IV. Ease of aggregation parallels functional activity; urea disaggregates and NaCl facilitates further aggregation of the peptides. Peptide acids aggregate only in media of high ionic strength. Hysteresis of the aggregation–disaggregation process is detected and ascribed to cooperativity of aggregation. The aggregation process is enthalpically favored, ?H ? ?1 to ?3 kcal mol?¹.

This data, together with the reported value of the dipole moment of alamethicin, has been utilized in Chapter VI to propose a model for the alamethicin channel consistent with a large body of published data. The model envisages aqueous?phase aggregation of hydrophobic ??helices in an antiparallel fashion, with three?fold symmetry followed by insertion into the bilayer. Changes in pore conductance states are brought about by changes in either the net dipole moment of the aggregate or the number of monomers in the aggregate, or both.

SECTION II describes studies on model systems, and a general introduction to membranes is presented in Chapter VII. Biological membranes are stabilized principally by hydrophobic interactions. Fluorescence probes have been used to investigate the structure of detergent micelles, liposomes, and bile salt micelles in Chapter VIII. Fluorescence intensity, wavelength of maximum emission, fluorescence depolarization, and quenching are consistent with a picture of the detergent micelle containing some water in its interior but not continuous with bulk water. SDS is more polar than CTAB and Triton?100. The hydrocarbon region of liposomes is much less polar than that of detergent micelles. The formation of primary and secondary micelles of bile salts can be detected by fluorescence probes. Dihydroxy acids form secondary micelles with more rigid hydrophobic associations than trihydroxy bile acids.

Chapter IX describes studies on model proteins. Tyrosyl fluorescence of serum albumins has been unmasked by binding bilirubin, which quenches tryptophyl fluorescence. Differences in the tyrosyl environment of human and bovine serum albumins are detected by fluorescence quenching. The sulfhydryl environments of the serum albumins, ovalbumin, and papain have been probed by covalently labeling with dansyl aziridine. The label detects the N–B transition of serum albumins and unfolding of the proteins by urea.

The major findings of this investigation are summarized in Chapter X, the General Summary.