Orientation and Membrane Partition Free Energy of PeT-Based Voltage-Sensitive Dyes from Molecular Simulations.

Voltage measurement via small-molecule fluorescent indicators is a valuable approach in deciphering complex dynamics in electrically excitable cells. However, our understanding of various physicochemical properties governing the performance of fluorescent voltage sensors based on the photoinduced electron transfer (PeT) mechanism remains incomplete. Here, through extensive molecular dynamics and free energy calculations, we systematically examine the orientation and membrane partition of three PeT-based voltage-sensing VoltageFluor (VF) dyes in different lipid environment. We show that the symmetry of the molecular scaffold and the net charge of the hydrophilic headgroup of a given VF dye dominate its orientation and membrane partition, respectively. Our work provides a mechanistic understanding of the physical properties contributing to the voltage sensitivity, signal-to-noise ratio, as well as membrane distribution of VF dyes and sheds light onto rational design principles of PeT-based fluorescent probes in general.

Ion Conduction Mechanisms in Potassium Channels Revealed by Permeation Cycles.

Potassium channels are responsible for the selective yet efficient permeation of potassium ions across cell membranes. Despite many available high-resolution structures of potassium channels, those conformations inform only on static information on the ion permeation processes. Here, we use molecular dynamics simulations and Markov state models to obtain dynamical details of ion permeation. The permeation cycles, expressed in terms of selectivity filter occupancy and representing ion permeation events, are illustrated. We show that the direct knock-on permeation represents the dominant permeation mechanism over a wide range of potassium concentrations, temperatures, and membrane voltages for the pore of MthK. Direct knock-on is also observed in other potassium channels with a highly conserved selectivity filter, demonstrating the robustness of the permeation mechanism. Lastly, we investigate the charge strength dependence of permeation cycles. Our results shed light on the underlying permeation details, which are valuable in studying conduction mechanisms in potassium channels.

Structural and dynamic basis of substrate permissiveness in hydroxycinnamoyltransferase (HCT).

Substrate permissiveness has long been regarded as the raw materials for the evolution of new enzymatic functions. In land plants, hydroxycinnamoyltransferase (HCT) is an essential enzyme of the phenylpropanoid metabolism. Although essential enzymes are normally associated with high substrate specificity, HCT can utilize a variety of non-native substrates. To examine the structural and dynamic basis of substrate permissiveness in this enzyme, we report the crystal structure of HCT from Selaginella moellendorffii and molecular dynamics (MD) simulations performed on five orthologous HCTs from several major lineages of land plants. Through altogether 17-µs MD simulations, we demonstrate the prevalent swing motion of an arginine handle on a submicrosecond timescale across all five HCTs, which plays a key role in native substrate recognition by these intrinsically promiscuous enzymes. Our simulations further reveal how a non-native substrate of HCT engages a binding site different from that of the native substrate and diffuses to reach the catalytic center and its co-substrate. By numerically solving the Smoluchowski equation, we show that the presence of such an alternative binding site, even when it is distant from the catalytic center, always increases the reaction rate of a given substrate. However, this increase is only significant for enzyme-substrate reactions heavily influenced by diffusion. In these cases, binding non-native substrates 'off-center' provides an effective rationale to develop substrate permissiveness while maintaining the native functions of promiscuous enzymes.