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

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

Charge Regulation during Amyloid Formation of α-Synuclein

[Image: see text] Electrostatic interactions play crucial roles in protein function. Measuring pK(a) value perturbations upon complex formation or self-assembly of e.g. amyloid fibrils gives valuable information about the effect of electrostatic interactions in those processes. Site-specific pK(a) value determination by solution NMR spectroscopy is challenged by the high molecular weight of amyloid fibrils. Here we report a pH increase during fibril formation of α-synuclein, observed using three complementary experimental methods: pH electrode measurements in water; colorimetric changes of a fluorescent indicator; and chemical shift changes for histidine residues using solution state NMR spectroscopy. A significant pH increase was detected during fibril formation in water, on average by 0.9 pH units from 5.6 to 6.5, showing that protons are taken up during fibril formation. The pH upshift was used to calculate the average change in the apparent pK(a)(ave) value of the acidic residues, which was found to increase by at least 1.1 unit due to fibril formation. Metropolis Monte Carlo simulations were performed on a comparable system that also showed a proton uptake due to fibril formation. Fibril formation moreover leads to a significant change in proton binding capacitance. Parallel studies of a mutant with five charge deletions in the C-terminal tail revealed a smaller pH increase due to fibril formation, and a smaller change (0.5 units on average) in the apparent pK(a)(ave) values of the acidic residues. We conclude that the proton uptake during the fibril formation is connected to the high density of acidic residues in the C-terminal tail of α-synuclein

Autocatalytic amplification of Alzheimer-associated Aβ42 peptide aggregation in human cerebrospinal fluid

Funder: Knut och Alice Wallenbergs Stiftelse (Knut and Alice Wallenberg Foundation); doi: https://doi.org/10.13039/501100004063Funder: Alzheimerfonden; doi: https://doi.org/10.13039/501100008599Abstract: Alzheimer’s disease is linked to amyloid β (Aβ) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aβ aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aβ42 in human CSF through kinetic experiments at several Aβ42 monomer concentrations (0.8–10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis

It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies. (c) 2006 Elsevier B.V. All rights reserved