Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy

Abstract

KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45–71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR structure of KCNE1 in micelles.

Biophysical EPR Studies Applied to Membrane Proteins

“…Unique structural and dynamic information on membrane proteins can be gleaned using these powerful techniques. Table 1 shows recent examples of some biologically important membrane protein systems studied by using EPR spectroscopy [43,44,49,64,74,75,78,79]. EPR spectroscopy has become complementary to existing biophysical methods to study membrane proteins.…”

“…Under experimental conditions, these motions can be isolated from the EPR spectrum. The inverse linewidth of the central line of the EPR spectrum provides a measure of relative mobility [9,43,44]. The inverse linewidth mobility against the amino acid sequence can produce a periodic data profile, which can be used to predict the local secondary structure of the proteins and peptides [9,10,45].…”

“…The inverse linewidth mobility against the amino acid sequence can produce a periodic data profile, which can be used to predict the local secondary structure of the proteins and peptides [9,10,45]. There are several biologically important membrane proteins (e.g., prokaryotic potassium channel KcsA, KCNE1, lactose permease protein, and KvAP voltage-sensing domain) that have used CW-EPR spectroscopy to probe their secondary structure [43,44,46,47]. This method can also be used to indentify functional domains in high molecular weight proteins, supramolecular complexes and membrane proteins [20].…”

Site-Directed Spin Labeling EPR for Studying Membrane Proteins

“…Under experimental conditions, these motions can be isolated from the EPR spectrum. The inverse linewidth of the central line of the EPR spectrum provides a measure of relative mobility [ 22 , 25 , 51 , 56 ]. A plot of the inverse linewidth mobility against the amino acid sequence can produce a periodic data profile, which can be used to predict the local secondary structure of the proteins and peptides [ 8 , 22 , 51 , 57 ].…”

“…In the solvent phase, a spin labeled peptide or small protein rapidly tumbling leads to an isotropic spectrum with a rotational correlation time of less than nanosecond. However, in a membrane, spin labeled peptides experience restricted mobility, resulting in a broader EPR spectrum with two motional components resulting from the superposition of the signals arising from a free and bound peptide [ 51 , 54 , 59 – 61 ]. Protein topology in a membrane can be studied with respect to the membrane using nitroxide based SDSL EPR power saturation experiments [ 8 , 60 – 62 ].…”