The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.

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-

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL–GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

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-

The organization and orientation of membrane-inserted helices is important for better understanding the mode of action of membrane-active peptides and of protein-membrane interactions. Here we report on the application of ESEEM (electron spin-echo envelope modulation) and DEER (double electron-electron resonance) techniques to probe the orientation and oligomeric state of an α-helical trans-membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin-labeled WALP23 and (2)H nuclei of either the solvent (D2O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of (2)H nuclei in the hydrophilic region of the membrane and the density of the (2)H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin-labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N-terminus is more buried than the C-terminus. In addition, the ESEEM data fitting yielded a (2)H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented.