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Structural characterization of the voltage-sensor domain and voltage-gated K+-channel proteins vectorially oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry.

ABSTRACT: The voltage-sensor domain (VSD) is a modular four-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of X-ray crystal structures for a few voltage-gated potassium (Kv) channels and a voltage-gate sodium (Nav) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e., nonconducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially oriented within a single phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) membrane investigated by X-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces, thus achieving partial to full hydration, respectively (Gupta et al. Phys. Rev. E2011, 84, 031911-1-15). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the submolecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected submolecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and exchangeable hydrogen throughout the profile structure of both the VSD itself and the VSD:POPC membrane. These two experimentally determined water and exchangeable hydrogen distribution profiles are in good agreement with molecular dynamics simulations of the VSD protein vectorially oriented within a fully hydrated POPC bilayer membrane, supporting the existence of the VSD's water pore. This approach was extended to the full-length Kv-channel (KvAP) at a solid/liquid interface, providing the separate profile structures of the KvAP protein and the POPC bilayer within the reconstituted KvAP:POPC membrane.

SUBMITTER: Gupta S

PROVIDER: S-EPMC3406608 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Structural characterization of the voltage-sensor domain and voltage-gated K+-channel proteins vectorially oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry.

Gupta S S Dura J A JA Freites J A JA Tobias D J DJ Blasie J K JK

Langmuir : the ACS journal of surfaces and colloids 20120629 28

The voltage-sensor domain (VSD) is a modular four-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of X-ray crystal structures for a few voltage-gated potassium (Kv) channels and a voltage-gate sodium (Nav) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e., nonconducting to ca ...[more]

PMID: 22686684

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Project description:One subunit of the prokaryotic voltage-gated potassium ion channel from Aeropyrum pernix (KvAP) is comprised of six transmembrane α helices, of which S1-S4 form the voltage-sensor domain (VSD) and S5 and S6 contribute to the pore domain (PD) of the functional homotetramer. However, the mechanism of electromechanical coupling interconverting the closed-to-open (i.e., nonconducting-to-K(+)-conducting) states remains undetermined. Here, we have vectorially oriented the detergent (OG)-solubilized VSD in single monolayers by two independent approaches, namely "directed-assembly" and "self-assembly," to achieve a high in-plane density. Both utilize Ni coordination chemistry to tether the protein to an alkylated inorganic surface via its C-terminal His_{6} tag. Subsequently, the detergent is replaced by phospholipid (POPC) via exchange, intended to reconstitute a phospholipid bilayer environment for the protein. X-ray interferometry, in which interference with a multilayer reference structure is used to both enhance and phase the specular x-ray reflectivity from the tethered single membrane, was used to determine directly the electron density profile structures of the VSD protein solvated by detergent versus phospholipid, and with either a moist He (moderate hydration) or bulk aqueous buffer (high hydration) environment to preserve a native structure conformation. Difference electron density profiles, with respect to the multilayer substrate itself, for the VSD-OG monolayer and VSD-POPC membranes at both the solid-vapor and solid-liquid interfaces, reveal the profile structures of the VSD protein dominating these profiles and further indicate a successful reconstitution of a lipid bilayer environment. The self-assembly approach was similarly extended to the intact full-length KvAP channel for comparison. The spatial extent and asymmetry in the profile structures of both proteins confirm their unidirectional vectorial orientation within the reconstituted membrane and indicate retention of the protein's folded three-dimensional tertiary structure upon completion of membrane bilayer reconstitution. Moreover, the resulting high in-plane density of vectorially oriented protein within a fully hydrated single phospholipid bilayer membrane at the solid-liquid interface will enable investigation of their conformational states as a function of the transmembrane electric potential.

Project description: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.

Dataset Information

Structural characterization of the voltage-sensor domain and voltage-gated K+-channel proteins vectorially oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry.

Publications

Structural characterization of the voltage-sensor domain and voltage-gated K+-channel proteins vectorially oriented within a single bilayer membrane at the solid/vapor and solid/liquid interfaces via neutron interferometry.

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