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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.

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 laevishave 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.

Human carbonic anhydrase IX (CAIX) has evolved as a promising biomarker for cancer prognosis, due to its overexpression in various cancers and restricted expression in normal tissue. However, limited information is available on its biophysical behavior. The unfolding of CAIX in aqueous urea solution was studied using all-atom molecular dynamics simulation approach. The results of this study revealed a stable intermediate state along the unfolding pathway of CAIX. At intermediate concentrations of urea (2.0–4.0 M), the protein displays a native-like structure with a large population of its secondary structure and hydrophobic contacts remaining intact in addition to small confined overall motions. Beyond 4.0 M urea, the unfolding is more gradual and at 8.0 M urea the structure is largely collapsed due to the solvent effect. The hydrophobic contact analysis suggests that the contact in terminal α-helices is separated initially which propagates in the loss of contacts from centrally located β-sheets. The reduction of 60–65% tertiary contacts in 7.0–8.0 M urea suggested the presence of residual structure in unfolded state and is confirmed with structural snap shot. Free energy landscape analysis suggested that unfolding of CAIX exists through the different intermediate states.