Porin in its' lipid environment

O'Keeffe, Aisling (2000) Porin in its' lipid environment. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Porins are a family of proteins found in the outer membranes of Gram negative bacteria. These proteins form water-filled transmembrane pores which allow the influx of nutrients into the bacterial cell. The pore structure is formed by a closed β-barrel giving a highly stable structure. Interactions of this group of β-barrel proteins with its surrounding lipid were investigated. A method for reconstituting the Escherichia coli OmpF porin into membrane fragments of defined lipid composition was developed and this method was used in fluorescence experiments with brominated phospholipids to assess the degree of lipid binding selectivity of the porins. Preferential binding was observed with phosphatidylcholines possessing fatty acyl chains with a length of 14 carbons. This is in contrast to α-helical proteins, such as the Ca²⁺ -ATPase which show little lipid binding selectivity because they are less rigid and can adapt to different bilayer thickness by tilting in the membrane. The fluorescence properties of the two tryptophan residues on OmpF were studied independently by using recombinant techniques to yield single tryptophan and tryptophan-less mutants. These provided information regarding the environment of the tryptophan residues. Quenching with spin-labelled fatty acids was also carried out to determine the accessibility of the interface-located tryptophan. Functional studies were also performed to determine the effects of hydrophobic mismatch on the permeability rates of sugars through OmpF, and it was seen that permeability rates of monosaccharide sugars through OmpF are highest in dioleylphosphatidylcholine.

Text

785678.pdf - Version of Record

Available under License University of Southampton Thesis Licence.

Download (16MB)