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

Abstract

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 (T_m) 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.

Figure 1.

(A) Chemical structure of the MTSL spin label probe, (B) predicted topology of Q1-VSD (100–249) in lipid bilayers based on previous solution NMR studies, and (C) chemical structure of 3:1 SMA polymer. Red circles represent the mutants linked to long QT syndrome.

Figure 2.

CW-EPR spectra of Q1-VSD bearing MTSL at F130C (A), V165C (B), and Q147C (C) in POPC/POPG liposomes (left) and POPC/POPG lipodisq nanoparticles (right) as a function of temperature. Arrows represent the two motional components in the spectra.

Figure 3.

Inverse central line width as a function of temperature for Q1-VSD bearing MTSLs at sites F130C (A), V165C (B), and Q147C(C) calculated from EPR spectra in Figure 2.

Figure 4.

CW-EPR spectral simulations of Q1-VSD mutants bearing MTSLs at 297 and 325 K at the sites F130C (A), V165C (B), and Q147C (C) using the NLSL MOMD program developed by Freed and co-workers.^,

Figure 5.

Q-band DEER data of Q1-VSD mutants (Phe123/Ser143) bearing two MTSL spin labels. Background-subtracted dipolar evolutions of the indicated mutants (left) and their corresponding distance probability distributions from Tikhonov regularization (right) for 1% LMPG micelles (A), 0.5% DPC micelles (B), proteoliposomes (POPC/POPG = 3:1) (C), bicelles (DMPC/DPC= 3.2:1) (D), and lipodisq nanoparticles (E).

Figure 6.

Experimental phase memory curves for dual-spin-labeled Q1-VSD mutants (Phe123/Ser143) bearing two MTSL spin labels for liposomes (POPC/POPG = 3:1) (A) and POPC/POPG lipodisq nanoparticles (B). T_m values are 1.2 ± 0.2 μs for proteoliposomes and 2.0 ± 0.2 μs for lipodisq nanoparticles.