The heart of the H ؉ conductance mechanism in the homotetrameric M2 H ؉ channel from influenza A is a set of four histidine side chains. Here, we show that protonation of the third of these imidazoles coincides with acid activation of this transmembrane channel and that, at physiological pH, the channel is closed by two imidazole-imidazolium dimers, each sharing a low-barrier hydrogen bond. This unique construct succeeds in distributing a pair of charges over four rings and many atoms in a low dielectric environment to minimize charge repulsion. These dimers form with identical pK as of 8.2 ؎ 0.2, suggesting cooperative H ؉ binding and clearly illustrating high H ؉ affinity for this channel. The protonation behavior of the histidine side chains has been characterized by using solid-state NMR spectroscopy on the M2 transmembrane domain in fully hydrated lipid bilayers where the tetrameric backbone structure is known. Furthermore, electrophysiological measurements of multichannel and single-channel experiments confirm that these protein constructs are functional.M2 channel ͉ proton channel ͉ solid-state NMR ͉ low-barrier hydrogen bond ͉ histidine ionization constants A histidine tetrad in the pore of the tetrameric M2 protein has long been associated with key channel features of H ϩ selectivity, pH activation, gating, inhibition, and the specific conductance mechanism. M2 protein from influenza A virus conducts protons into the viral core after endocytosis, which leads to the uncoating and release of genetic material into the cytoplasm after fusion of the viral coat with the endosomal wall (1, 2). Much is known about this system from its tetrameric state (2-4), the backbone structure of the transmembrane (TM) domain (5), and numerous electrophysiological (6, 7), biophysical (8-10), and modeling (11) studies that have cast a fascinating tale for this important influenza drug target and the only proton channel of its kind to be characterized in such detail. However, the specific role of His-37 in the tetrameric protein has not been elucidated. Here, we have characterized the pK a s associated with this cluster of four histidine residues in the hydrophobic interstices of the membrane. These pK a values have led us to substantial mechanistic conclusions.There are many lines of evidence, reviewed by Kelly et al. (6), that support the conclusion that M2 is responsible for viral acidification. In vivo ion conductance recordings have shown pH sensitive conductance resulting in rapid acidification of the Xenopus oocytes (12, 13) and mammalian cells (13-15) containing M2 protein.Preparations of purified M2 protein have also been used to show proton conductance in synthetic lipid bilayers (16,17). Singlechannel conductance measurements with membranes containing M2 protein give clear evidence that it is H ϩ conductance, not counterion conductance, that is observed. Furthermore, the channel conductance is unchanged by addition of an excess of NaCl (18). Conductance measurements for the isolated TM domain of M2 protein have als...

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins, or alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change not only affects thermodynamic properties but also dynamic properties, including the amplitudes of motions on different timescales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, e.g., the relation between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

The contributions of electrostatic interactions to the binding stability of barnase and barstar were studied by the Poisson-Boltzmann model with three different protocols: a), the dielectric boundary specified as the van der Waals (vdW) surface of the protein along with a protein dielectric constant (epsilon (p)) of 4; b), the dielectric boundary specified as the molecular (i.e., solvent-exclusion (SE)) surface along with epsilon (p) = 4; and c), "SE + epsilon (p) = 20." The "vdW + epsilon (p) = 4" and "SE + epsilon (p) = 20" protocols predicted an overall electrostatic stabilization whereas the "SE + epsilon (p) = 4" protocol predicted an overall electrostatic destabilization. The "vdW + epsilon (p) = 4" protocol was most consistent with experiment. It quantitatively reproduced the observed effects of 17 mutations neutralizing charged residues lining the binding interface and the measured coupling energies of six charge pairs across the interface and reasonably rationalized the experimental ionic strength and pH dependences of the binding constant. In contrast, the "SE + epsilon (p) = 4" protocol predicted significantly larger coupling energies of charge pairs whereas the "SE + epsilon (p) = 20" protocol did not predict any pH dependence. This study calls for further scrutiny of the different Poisson-Boltzmann protocols and demonstrates potential danger in drawing conclusions on electrostatic contributions based on a particular calculation protocol.

Membraneless organelles, corresponding to the droplet phase upon liquid–liquid phase separation (LLPS) of protein or protein–RNA mixtures, mediate myriad cellular functions. Cells use a variety of biochemical signals such as expression level and posttranslational modification to regulate droplet formation and dissolution, but the physical basis of the regulatory mechanisms remains ill-defined and quantitative assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromolecular regulators promote or suppress LLPS. We experimentally tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH35 and PRM5) as droplet-forming proteins. Determination of the changes in phase boundary and the partition coefficients in the droplet phase over a wide range of regulator concentrations yielded both a quantitative measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were observed. Ficoll 70 at high concentrations indirectly promoted SH35–PRM5 LLPS, by taking up volume in the bulk phase and thereby displacing SH35 and PRM5 into the droplet phase. Lysozyme had a moderate partition coefficient and suppressed LLPS by substituting weaker attraction with SH35 for the stronger SH35–PRM5 attraction in the droplet phase. By forming even stronger attraction with PRM5, heparin at low concentrations partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromolecular regulators as volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.