Probing Proton Translocation in Influenza A/M2 Proteoliposomes - A systematic Approach to Membrane-Protein Reconstitutions

Research output: ThesisDoctoral Thesis (compilation)

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Abstract

An improved method for reconstituting membrane proteins into artificial liposomes for quantitative
functional analysis is presented. A number of key parameters for reconstitution by detergent removal are
assessed in this thesis: The lipid-to-protein ratio, the detergent-to-lipid ratio and the lipid and cholesterol
composition. New porphyrin-based pH-probes are evaluated. Based on this systematic, comprehensive
approach to protein reconstitution, we present a robust system for quantitative proton-flux analysis, as
demonstrated by influenza virus A M2 reconstitution into large unilamellar vesicles.

The M2 protein is a small, single-span transmembrane protein, which plays an important role in the
life cycle of influenza A virus and is the target of the adamantane series of anti-influenza drugs. This virus
enters cells via the endosomes; as the endosomes acidify M2 facilitates proton transport into the viral
interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been
how protons can accumulate in the viral interior without developing a large electrical potential that
impedes further inward proton translocation, which is required to effect a significant change in the
internal effective pH. Here, we show that M2 has essential antiporter-like activity. This should lead to
future investigations of the biophysical mechanism of transport, which will have implications for the
design of new generations of M2-targeting drugs as well as furthering our understanding of cotransport.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biochemistry and Structural Biology
Supervisors/Advisors
  • Peterson Årsköld, Sindra, Supervisor
Award date2010 May 28
Publication statusPublished - 2010

Bibliographical note

Defence details

Date: 2010-05-28
Time: 09:30
Place: Kemicentrum hörsal C

External reviewer(s)

Name: Moser, Christopher
Title: Professor
Affiliation: University of Pennsylvania, USA

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Subject classification (UKÄ)

  • Biological Sciences

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