The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic 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. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K þ or Na þ efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K þ to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH in ) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH in first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH in is achieved.antiporter | liposomes | M2 channel | protons | ion channel T he influenza A virus M2 protein is a tetrameric integral membrane protein containing a short N-terminal extracellular domain, a transmembrane (TM) helix, and a 54-residue cytoplasmic tail (1, 2). Four M2 TM domains associate into a highly selective proton channel (3, 4) whose activity is essential for viral replication (5, 6). Influenza virus enters cells via the endosomal pathway, and the M2 protein functions to equilibrate the pH of the virus interior with that of the acidic endosome. The lowering of the pH within the virus leads to disruption of the interactions between the viral ribonucleoprotein (RNP) complex and the M1 protein (5), an important step in viral uncoating (7). Additionally, for some subtypes of influenza A virus, the M2 proton channel activity helps maintain a neutral pH in the lumen of the trans-Golgi network to prevent premature triggering of the hemagglutinin to the low-pH form (8-11). A long-standing question has been how M2 can conduct a substantial number of protons into the very small-volume interior of the virus without developing a substantial electrical gradient. Diffusion of a few protons down their concentration gradient from the acid environment of the endosome into the viral interior would result in an electrical gradient that would abruptly halt further proton flow and thereby prevent acidification of the viral interior. Here we...

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.

The nucleation of Alzheimer-associated Aβ peptide monomers can be catalyzed by preexisting Aβ fibrils. This leads to autocatalytic amplification of aggregate mass and underlies self-replication and generation of toxic oligomers associated with several neurodegenerative diseases. However, the nature of the interactions between the monomeric species and the fibrils during this key process, and indeed the ultrastructural localization of the interaction sites have remained elusive. Here we used NMR and optical spectroscopy to identify conditions that enable the capture of transient species during the aggregation and secondary nucleation of the Aβ42 peptide. Cryo-electron microscopy (cryo-EM) images show that new aggregates protrude from the entire length of the progenitor fibril. These protrusions are morphologically distinct from the well-ordered fibrils dominating at the end of the aggregation process. The data provide direct evidence that self-replication through secondary nucleation occurs along the sides of fibrils, which become heavily decorated under the current solution conditions (14 µM Aβ42, 20 mM sodium phosphate, 200 µM EDTA, pH 6.8).

Metal-free porphyrin-dendrimers provide a convenient platform for construction of membraneimpermeable ratiometric probes for pH measurements in compartmentalized biological systems. In all previously reported molecules, electrostatic stabilization (shielding) of the core porphyrin by peripheral negative charges (carboxylates) was required to shift the intrinsically low porphyrin protonation pK a 's into the physiological pH range (pH 6-8). However, binding of metal cations (e.g. K + , Na + , Ca 2+ , Mg 2+ ) by the carboxylate groups on the dendrimer could affect the protonation behavior of such probes in biological environments. Here we present a dendritic pH nanoprobe based on a highly non-planar tetraaryltetracyclohexenoporphyrin (Ar 4 TCHP), whose intrinsic protonation pK a 's are significantly higher than those of regular tetraarylporphyrins, thereby eliminating the need for electrostatic core shielding. The porphyrin was modified with eight Newkome-type dendrons and PEGylated at the periphery, rendering a neutral water-soluble probe (TCHpH), suitable for measurements in the physiological pH range. The protonation of TCHpH could be followed by absorption (e.g. ε Soret (dication)~270,000 M −1 cm −1 ) or by fluorescence. Unlike most tetraarylporphyrins, TCHpH is protonated in two distinct steps (pK a 's 7.8 and 6.0). In the region between the pK a 's, an intermediate species with a well-defined spectroscopic signature, presumably a TCHpH monocation, could be observed in the mixture. The performance of TCHpH was evaluated by pH gradient measurements in large unilamellar vesicles. The probe was retained inside the vesicles and did not pass through and/or interact with vesicle membranes, proving useful for quantification of proton transport across phospholipid bilayers. To interpret the protonation behavior of TCHpH we developed a model relating structural changes on the porphyrin macrocycle upon protonation to its basicity. The model was validated by DFT calculations performed on a planar and non-planar porphyrin, making it possible to rationalize higher protonation pK a 's of non-planar porphyrins as well as the easier observation of their monocations.

Electrostatic interactions play crucial roles in protein function. Measuring p K 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 p K 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 p K 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 p K 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.