Purification and membrane interactions of human KCNQ1100-370 potassium ion channel.

KCNQ1 (Kv7.1 or KvLQT1) is a voltage-gated potassium ion channel that is involved in the ventricular repolarization following an action potential in the heart. It forms a complex with KCNE1 in the heart and is the pore forming subunit of slow delayed rectifier potassium current (Iks). Mutations in KCNQ1, leading to a dysfunctional channel or loss of activity have been implicated in a cardiac disorder, long QT syndrome. In this study, we report the overexpression, purification, biochemical characterization of human KCNQ1100-370, and lipid bilayer dynamics upon interaction with KCNQ1100-370. The recombinant human KCNQ1 was expressed in Escherichia coli and purified into n-dodecylphosphocholine (DPC) micelles. The purified KCNQ1100-370 was biochemically characterized by SDS-PAGE electrophoresis, western blot and nano-LC-MS/MS to confirm the identity of the protein. Circular dichroism (CD) spectroscopy was utilized to confirm the secondary structure of purified protein in vesicles. Furthermore, 31P and 2H solid-state NMR spectroscopy in DPPC/POPC/POPG vesicles (MLVs) indicated a direct interaction between KCNQ100-370 and the phospholipid head groups. Finally, a visual inspection of KCNQ1100-370 incorporated into MLVs was confirmed by transmission electron microscopy (TEM). The findings of this study provide avenues for future structural studies of the human KCNQ1 ion channel to have an in depth understanding of its structure-function relationship.

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State.

NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1-300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

Characterization of the Human KCNQ1 Voltage Sensing Domain (VSD) in Lipodisq Nanoparticles for Electron Paramagnetic Resonance (EPR) Spectroscopic Studies of Membrane Proteins.

Membrane proteins are responsible for conducting essential biological functions that are necessary for the survival of living organisms. In spite of their physiological importance, limited structural information is currently available as a result of challenges in applying biophysical techniques for studying these protein systems. Electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study the structural and dynamic properties of membrane proteins. However, the application of EPR spectroscopy to membrane proteins in a native membrane-bound state is extremely challenging due to the complexity observed in inhomogeneity sample preparation and the dynamic motion of the spin label. Detergent micelles are very popular membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is important to test whether the protein structure in a micelle environment is the same as that of its membrane-bound state. Lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have been introduced as a potentially good membrane-mimetic system for structural studies of membrane proteins. Recently, we reported on the EPR characterization of the KCNE1 membrane protein having a single transmembrane incorporated into lipodisq nanoparticles. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the more complicated membrane protein system human KCNQ1 voltage sensing domain (Q1-VSD) having four transmembrane helices using site-directed spin-labeling EPR spectroscopy. Characterization of spin-labeled Q1-VSD incorporated into lipodisq nanoparticles was carried out using CW-EPR spectral line shape analysis and pulsed EPR double-electron electron resonance (DEER) measurements. The CW-EPR spectra indicate an increase in spectral line broadening with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated a superior quality of distance measurement with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles when compared to proteoliposomes. These results are consistent with the solution NMR structural studies on the Q1-VSD. This study will be beneficial for researchers working on investigating the structural and dynamic properties of more complicated membrane protein systems using lipodisq nanoparticles.

Simple Derivatization of RAFT-Synthesized Styrene-Maleic Anhydride Copolymers for Lipid Disk Formulations.

Styrene-maleic acid copolymers have received significant attention because of their ability to interact with lipid bilayers and form styrene-maleic acid copolymer lipid nanoparticles (SMALPs). However, these SMALPs are limited in their chemical diversity, with only phenyl and carboxylic acid functional groups, resulting in limitations because of sensitivity to low pH and high concentrations of divalent metals. To address this limitation, various nucleophiles were reacted with the anhydride unit of well-defined styrene-maleic anhydride copolymers in order to assess the potential for a new lipid disk nanoparticle-forming species. These styrene-maleic anhydride copolymer derivatives (SMADs) can form styrene-maleic acid derivative lipid nanoparticles (SMADLPs) when they interact with lipid molecules. Polymers were synthesized, purified, characterized by Fourier-transform infrared spectroscopy, gel permeation chromatography, and nuclear magnetic resonance and then used to make disk-like SMADLPs, whose sizes were measured by dynamic light scattering (DLS). The SMADs form lipid nanoparticles, observable by DLS and transmission electron microscopy, and were used to reconstitute a spin-labeled transmembrane protein, KCNE1. The polymer method reported here is facile and scalable and results in functional and robust polymers capable of forming lipid nanodisks that are stable against a wide pH range and 100 mM magnesium.

Probing the Dynamics and Structural Topology of the Reconstituted Human KCNQ1 Voltage Sensor Domain (Q1-VSD) in Lipid Bilayers Using Electron Paramagnetic Resonance Spectroscopy.

KCNQ1 (Kv7.1 or KvLQT1) is a potassium ion channel protein found in the heart, ear, and other tissues. In complex with the KCNE1 accessory protein, it plays a role during the repolarization phase of the cardiac action potential. Mutations in the channel have been associated with several diseases, including congenital deafness and long QT syndrome. Nuclear magnetic resonance (NMR) structural studies in detergent micelles and a cryo-electron microscopy structure of KCNQ1 from Xenopus laevis have shown that the voltage sensor domain (Q1-VSD) of the channel has four transmembrane helices, S1-S4, being overall structurally similar with other VSDs. In this study, we describe a reliable method for the reconstitution of Q1-VSD into (POPC/POPG) lipid bilayer vesicles. Site-directed spin labeling electron paramagnetic resonance spectroscopy was used to probe the structural dynamics and topology of several residues of Q1-VSD in POPC/POPG lipid bilayer vesicles. Several mutants were probed to determine their location and corresponding immersion depth (in angstroms) with respect to the membrane. The dynamics of the bilayer vesicles upon incorporation of Q1-VSD were studied using 31P solid-state NMR spectroscopy by varying the protein:lipid molar ratios confirming the interaction of the protein with the bilayer vesicles. Circular dichroism spectroscopic data showed that the α-helical content of Q1-VSD is higher for the protein reconstituted in vesicles than in previous studies using DPC detergent micelles. This study provides insight into the structural topology and dynamics of Q1-VSD reconstituted in a lipid bilayer environment, forming the basis for more advanced structural and functional studies.

Characterizing the structure of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for membrane protein spectroscopic studies.

Membrane proteins play an important role in maintaining the structure and physiology of an organism. Despite their significance, spectroscopic studies involving membrane proteins remain challenging due to the difficulties in mimicking their native lipid bilayer environment. Membrane mimetic systems such as detergent micelles, liposomes, bicelles, nanodiscs, lipodisqs have improved the solubility and folding properties of the membrane proteins for structural studies, however, each mimetic system suffers from its own limitations. In this study, using three different lipid environments, vesicles were titrated with styrene-maleic acid (StMA) copolymer leading to a homogeneous SMALP system (∼10 nm) at a weight ratio of 1:1.5 (vesicle: StMA solution). A combination of Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) was used to characterize these SMALPs. We used a controlled synthesis mechanism to synthesize StMA based block copolymers called reversible addition-fragmentation chain transfer polymerization (RAFT) SMALPs. Incorporation of the Voltage Sensor Domain of KCNQ1 (Q1-VSD) into RAFT SMALPs indicates that this is a promising application of this system to study membrane proteins using different biophysical techniques. V165C in Q1-VSD corresponding to the hydrophobic region was incorporated into the SMALP system. Continuous Wave-Electron Paramagnetic Resonance (CW-EPR) line shape analysis showed line shape broadening, exposing a lower rigid component and a faster component of the spin label.