Direct evidence of conformational changes associated with voltage gating in a voltage sensor protein by time-resolved X-ray/neutron interferometry.

The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD's profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure

Direct evidence of conformational changes associated with voltage gating in a voltage sensor protein by time-resolved X-ray/neutron interferometry.

The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD's profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure

Voltage-Dependent Profile Structures of a Kv-Channel via Time-Resolved Neutron Interferometry

Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, I: Structural Investigations via X-Ray Reflectivity from Langmuir Monolayers

We previously reported the synthesis and structural characterization of a model membrane protein comprised of an amphiphilic 4-helix bundle peptide with a hydrophobic domain based on a synthetic ion channel and a hydrophilic domain with designed cavities for binding the general anesthetic halothane. In this work, we synthesized an improved version of this halothane-binding amphiphilic peptide with only a single cavity and an otherwise identical control peptide with no such cavity, and applied x-ray reflectivity to monolayers of these peptides to probe the distribution of halothane along the length of the core of the 4-helix bundle as a function of the concentration of halothane. At the moderate concentrations achieved in this study, approximately three molecules of halothane were found to be localized within a broad symmetric unimodal distribution centered about the designed cavity. At the lowest concentration achieved, of approximately one molecule per bundle, the halothane distribution became narrower and more peaked due to a component of ∼19Å width centered about the designed cavity. At higher concentrations, approximately six to seven molecules were found to be uniformly distributed along the length of the bundle, corresponding to approximately one molecule per heptad. Monolayers of the control peptide showed only the latter behavior, namely a uniform distribution along the length of the bundle irrespective of the halothane concentration over this range. The results provide insight into the nature of such weak binding when the dissociation constant is in the mM regime, relevant for clinical applications of anesthesia. They also demonstrate the suitability of both the model system and the experimental technique for additional work on the mechanism of general anesthesia, some of it presented in the companion parts II and III under this title

Direct Evidence of Conformational Changes Associated with Voltage Gating in a Voltage Sensor Protein by Time-Resolved X‑ray/Neutron Interferometry

The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na⁺, K⁺) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD’s profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD’s atomic-level 3-D structure

Monolayers of a Model Anesthetic-Binding Membrane Protein: Formation, Characterization, and Halothane-Binding Affinity

hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The α-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide