DEER EPR Measurements for Membrane Protein Structures via Bifunctional Spin Labels and Lipodisq Nanoparticles

Abstract

Pulsed EPR DEER structural studies of membrane proteins in a lipid bilayer have often been hindered by difficulties in extracting accurate distances when compared to globular proteins. In this study, we employed a combination of three recently developed methodologies: 1) bi-functional spin labels (BSL), 2) SMA-Lipodisq nanoparticles, and 3) Q-band pulsed EPR measurements to obtain improved signal sensitivity, increased transverse relaxation time, and more accurate and precise distances in DEER measurements on the integral membrane protein KCNE1. The KCNE1 EPR data indicated ~2 fold increase in the transverse relaxation time for the SMA-Lipodisq nanoparticles when compared to proteoliposomes, and narrower distance distributions for the BSL when compared to the standard MTSL. The certainty of information content in DEER data obtained for KCNE1 in SMA-Lipodisq nanoparticles is comparable to that in micelles. The combination of techniques will enable researchers to potentially obtain more precise distances in cases where the traditional spin labels and membrane systems yield imprecise distance distributions.

High-resolution helix orientation in actin-bound myosin determined with a bifunctional spin label

“…This missing information means that a single labeling site on a helix cannot completely define how that helix is oriented within each myosin head, in a way that would allow direct modeling de novo. Such ambiguity should be alleviated by combining this approach with distance measurements between pairs of sites, detected by dipolar spin-spin interaction (24,45). Such an approach would be essentially equivalent to the method used in structure determination by NMR, where both orientation and distance constraints are combined (46).…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…Structure and structure-function relationships of macromolecules are areas of intense EPR effort [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17].…”

“…Coupled with site-directed spin-labeling (SDSL), EPR is oftentimes used to characterize protein and nucleic acid structures and dynamics, conformational changes, molecule folding, macromolecule complexes, and oligomeric structures [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15]17]. The majority of biomolecules do not contain unpaired electrons from which one can obtain an EPR signal; therefore, spin-labeling approaches have been developed [15,[18][19][20][21][22][23][24][25] where site-specific persistent radicals or paramagnetic metal-probes are incorporated at specific locations within a biomolecule.…”

The Styrene-Maleic Acid Copolymer: A Versatile Tool in Membrane Research

“…However, it is also possible to insert membrane proteins into SMALPs, as was initially shown for PagP and bacteriorhodopsin (Knowles et al 2009;Orwick-Rydmark et al 2012) and later for the potassium channel modulator protein KCNE1 (Sahu et al 2013). To achieve this, proteins can either first be solubilized with detergent and then reconstituted into liposomes by conventional techniques or native membranes containing the protein can be supplemented with synthetic lipid after which SMA is added to obtain SMALPs (see e.g., Goddard et al 2015).…”

“…Another technique that has been successfully used to characterize membrane proteins in SMALPs is EPR spectroscopy (Orwick-Rydmark et al 2012;Sahu et al 2013Sahu et al , 2014. This allowed for accurate distance measurements that revealed similar dynamics of bacteriorhodopsin as compared to its state in the plasma membrane (OrwickRydmark et al 2012).…”