Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics

Abstract

During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated.

The cation diffusion facilitator protein MamM’s cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

“…To probe divalent metal binding and the associated conformational changes reflected by intramolecular distance, site-directed spin labelling (SDSL) was employed in combination with both continuous-wave electron paramagnetic resonance (cw-EPR) and pulsed electron-electron double resonance (PELDOR) spectroscopic studies . These methods provide a wealth of dynamic and structural information, via observation both of changes in the local environment of an attached exogenous spin label elucidated by cw-EPR, and the determination of long-range (typically between 2-8 nm) distance measurements and distance distributions afforded by PELDOR spectroscopy (Mullen et al, 2016). For SDSL, a stable paramagnetic (1-oxyl-2,2,5,5-tetramethylpyrrolidin-3-yl)methylthiosulfonate spin label (MTSL) reacts specifically with cysteine residues present within the protein, hence allowing EPR to report on the protein via these specifically labelled sites.…”

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

“…To probe divalent metal binding and the associated conformational changes reflected by intra-molecular distance, site-directed spin labeling was employed in combination with both continuous-wave EPR (cw-EPR) and pulsed electron-electron double resonance (PELDOR) spectroscopic studies ( 21 ). These methods provide a wealth of dynamic and structural information via observations of both changes in the local environment of an attached exogenous spin label elucidated by cw-EPR and the determination of long-range (typically between 2–8 nm) distance measurements and distance distributions afforded by PELDOR spectroscopy ( 26 ). We have previously reported ( 21 ) that using only one of the two intrinsic MamM CTD cysteine residues simplifies EPR measurements; hence the studies reported here use the same C267S variant of MamM CTD, in which only the Cys-275 site is available for labeling.…”

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

“…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].…”