Gadolinium(III) Spin Labels for High‐Sensitivity Distance Measurements in Transmembrane Helices

Abstract

Distance determination, by pulse EPR techniques, between two spin labels attached to biomolecules has become an attractive methodology to probe conformations and assemblies of biomolecules in frozen solutions. [1][2][3] Among these techniques, double electron-electron resonance (DEER or PELDOR), [4,5] which can access distances in the range of 1.7 to 8 nm, is highly popular, [6][7][8] and the most widely used spin labels are nitroxide radicals. Membrane proteins in their natural environment are of particular interest for DEER applications, since those pose a considerable challenge for Xray crystallography and NMR spectroscopy. DEER studies of peptides and proteins in either reconstituted or model membranes [9][10][11] are considerably more challenging than those in solution, because the high local concentration of the spins in the membrane decreases the phase memory time and, therefore, sensitivity. [12] Most DEER measurements on nitroxide-labeled biomolecules are carried out at X-band frequencies (9.5 GHz, 0.35 T), and recently such measurements were demonstrated in frozen cells. [13,14] A major difficulty of such measurements is the reduction of nitroxides in the cell, which severely limits the scope of such exciting developments.Recently, Gd 3+ (S = 7/2) spin labels have been suggested as an alternative to nitroxide spin labels for W-band and Q-

Gd(III)–Gd(III) distance measurements with chirp pump pulses

“…Modifications of this approach involve the use of non-identical spin labels, in particular a Gd(III) center and a nitroxide [3,4], as well as different experimental schemes, such as relaxation-induced dipolar modulation (RIDME) [5] or continuous-wave EPR [6]. Distance measurements involving Gd spin labels have been successfully demonstrated on a number of systems, including model compounds, peptides, nanoparticles, proteins and DNAs [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Moreover, a promising biochemical property of Gd spin labels is the stability under reducing conditions, which recently enabled Gd-Gd distance measurements of peptides and proteins embedded in cellular environments [22,23].…”

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

“…-Gd 3? PELDOR distance measurements, simultaneous two-site attachment is required and has been established using chemical tags (Raitsimring et al 2007;Potapov et al 2010;Gordon-Grossman et al 2011;Lueders et al 2011;Song et al 2011;Yagi et al 2011;Garbuio et al 2013;Matalon et al 2013).…”

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

“…Recent work in a diverse collection of systems (Kaminker et al, 2012;Lueders et al, 2011;Matalon et al, 2013a;Song et al, 2011;Yulikov et al, 2012) has demonstrated that Gd 3+ offers many advantages as an alternative spin label for high-field DEER measurements (Goldfarb, 2012(Goldfarb, , 2014Potapov et al, 2010b;Raitsimring et al, 2007;Yagi et al, 2011). This manuscript extends the capabilities of Gd 3+ to include accurately resolving distances between multiple neighboring proteins within a membrane protein oligomer by leveraging the reduction of multispin effects.…”