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. Among these techniques, double electron-electron resonance (DEER or PELDOR), which can access distances in the range of 1.7 to 8 nm, is highly popular, 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 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. 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. 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 (S= 7/2) spin labels have been suggested as an alternative to nitroxide spin labels for W-band and Qband DEER distance measurements. Gd tags can be attached to proteins, similar to nitroxides, by site-directed spin labeling (SDSL). Gd features high sensitivity at high frequencies and the DEER measurements are free of orientation selection effects so that the distance distribution can readily be extracted from a single DEER measurement. Moreover, in the context of future development of in-cell DEER measurements, Gd chelates are stable under in vivo conditions as known from their applications as contrast agents for magnetic resonance imaging (MRI). Gd-Gd DEER has been demonstrated on model systems, proteins, peptides, and DNA, all in isotropic membrane-free solutions. Distance measurements between a Gd label and a nitroxide label have also been shown to yield attractive sensitivity. In this work, we continue to develop the approach of Gd–Gd DEER distance measurements and demonstrate for the first time such measurements in a model membrane. The model system we chose consists of the well-studied transmembrane helical WALP peptides in 1,2-dioleoyl-snglycero-3-phosphocholine (DOPC) vesicles. We demonstrate the sensitivity of W-band Gd–Gd DEER to small distance variations in a membrane. Using WALP peptides of different lengths we show that such measurements pick up, in addition to the helix extension, also subtle “cis–trans” effects arising from different positions of the labels with respect to the helix axes. In addition, we report the effect of the spin label interaction with the membranes on the measured distance distribution. We compared W-band DEER on WALP23 labeled with two different Gd tags with X-band DEER onWALP23 labeled with nitroxide tags. Here we used X-band, rather than W-band, to avoid complications owing to orientation selection. The differences observed are important and suggest that by employing different spin labels such effects can be isolated. Finally, we show that the effect of hydrophobic mismatch between peptide and membrane can be explored by Gd–Gd DEER. WALP23 was labeled at the N and C termini (see Table 1) with two nitroxides (WAL23-NO) using (l-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl)methanesulfonate (MTSSL) and two different Gd-DOTA derivatives, shown in Figure 1. WALP23-DOTA is labeled with a DOTA chelate and WALP23-C1 with DOTA with phenylethylamine substituents. The bulky substituents were designed to restrict the flexibility of the tag. The hydrophobic length of WALP23 (ca. 2.6 nm) is very close to that of the hydrophobic thickness of DOPC bilayers (ca. 2.7 nm). The sample composition was 50 mm WALP23 in DOPC multilamellar vesicles (MLV; 1:1000 peptide/lipid molar ratio). Details of the sample preparation are given in the Supporting Information. [*] Dr. E. Matalon, Dr. A. Feintuch, Prof. D. Goldfarb Department of Chemical Physics, Weizmann Institute of Science Rehovot, 76100 (Israel) E-mail: Daniella.goldfarb@weizmann.ac.il