Doubly spin-labeled nanodiscs to improve structural determination of membrane proteins by ESR

Abstract

Spin-labeled nanodiscs improve DEER distance measurement of membrane proteins.

Electron Paramagnetic Resonance as a Tool for Studying Membrane Proteins

“…Excellent work has been done in recent years to minimize these limitations. Sample preparations for the reconstitution of membrane proteins have been optimized in the presence of unlabeled proteins, bicelles, nanodics, lipodisq nanoparticles, a low protein/lipid molar ratio, and restricted spin label probes [44,136,147,[149][150][151][152][153][154][155][156]. Using deuterated protein and solvents can also enhance the phase memory times that contribute towards the improvement of data quality [56].…”

Probing Structural Dynamics of Membrane Proteins Using Electron Paramagnetic Resonance Spectroscopic Techniques

“…Technical and methodological developments have been recently enhanced in the structural biology field to minimize these limitations to obtain superior DEER data quality for membrane proteins. The reconstitution of membrane proteins has been optimized in the presence of unlabeled proteins, bicelles, nanodics, lipodisq nanoparticles, low protein/lipid molar ratio and restricted spin label probes [11,32,41,[160][161][162][163][164][165][166][167]. Using deuterated protein and solvents can also enhance the phase memory times that contribute towards the improvement of signal/noise and the data quality [111].…”

“…Nitroxide based SDSL DEER spectroscopy has been applied to study several membrane protein systems including KvAP voltage-sensing domain, pentameric ligand-gated channel, E. coli integral membrane sulfurtransferase (YgaP), bacteriorhodopsin, KCNE1, KCNE3, C99 amyloid precursor protein, human dihydroorotate dehydrogenase enzyme (HsDHODH), influenza A M2 protein, outer membrane cobalamin transporter BtuB in intact E. coli, cardiac Na + /Ca 2+ exchange (NCX1.1) protein, Na + /Proline transporter PutP Escherichia coli, tetrameric potassium ion channel KcsA, α-synuclein, membranefusion K/E peptides, ABC transporter MsbA, HCN channels, YetJ membrane protein, ectodomain of gp41, multimeric membrane transport proteins, and multidrug transporter LmrP [32,78,150,[161][162][163][167][168][169][170][171][172][173][174][175][176][177][178][179][180][181][182][183][184][185][186].…”

Identifying Protein Conformational Dynamics Using Spin‐label ESR

“…[9] It includes at least three ESR-based distance measurement techniques, double electron-electron resonance (DEER or PELDOR), doubleq uantum coherence (DQC), and relaxation-induced dipolar modulation enhancement (RIDME), all of which have recently emerged as the methodo fc hoice in the fields of biophysicsa sw ell as molecular and structuralb iology. [7,8,10] They are now routinelyu sed to characterize the conformation ensemble of proteins,w hich is associated with protein dynamics, [4,11] to reveal structural transitions betweendifferentconformations of membrane protein in liposomes or nanodiscs, [12][13][14] and to validate crystal structures in membrane environments. [15] To perform pulsed dipolar measurements, ad oubly spin-labeled protein is generally required and is prepared by the SDSL methodology ( Figure 1A).…”