Analysis of Light-Induced Conformational Changes of Natronomonas pharaonis Sensory Rhodopsin II by Time Resolved Electron Paramagnetic Resonance Spectroscopy † Enrica Bordignon 1 , Johann P. Klare 1,2 , Julia Holterhues 1 , Swetlana Martell 2 , Aliaksei Krasnaberski 1 , Martin Engelhard 2 and Heinz-Ju ¨ rgen Steinhoff* 1 1 Fachbereich Physik, Universita ¨ t Osnabru ¨ ck, Osnabru ¨ ck, Germany 2 Max-Planck-Institut fu ¨ r Molekulare Physiologie, Dortmund, Germany Received 5 July 2006; accepted 29 August 2006; published online 7 September 2006 DOI: 10.1562 ⁄ 2006-07-05-RA-960 ABSTRACT The nature and kinetics of the conformational changes leading to the activated state of NpSRII ⁄ NpHtrII 157 were investigated by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy in combination with site-directed spin labeling (SDSL) on a series of spin labeled mutants of NpSRII. A structural rearrangement of the cytoplasmic moiety of NpSRII upon light activation was detected (helices B, C, F and G). The increase in distance between helices C and F in the M-trapped state of the complex observed in one double mutant is in line with the notion that an outward movement of helix F occurs upon receptor activation. The data obtained from the NpSRII ⁄ NpH- trII 157 complex reconstituted in purple membrane lipids are compared with those obtained from the X-ray structure of the late M-state of the complex which shows some discrepancies. The results are discussed in the context also of other biophysical and EPR experimental evidences. INTRODUCTION In halophilic archaea like Halobacterium salinarum and Natronomonas pharaonis (Np), the photophobic response to green-blue light is mediated by sensory rhodopsin II (SRII, also named phoborhodopsin) (for recent reviews see [1–5]). SRII is structurally and functionally closely related to the light driven ion pumps bacteriorhodopsin (BR) and halorhodopsin (HR). These proteins comprise seven transmembrane helices (A–G) and a retinal chromophore covalently bound via a protonated Schiff base to a conserved lysine residue on helix G. The signal transduction to the intracellular two-component pathway, which modulates the swimming behavior of the cell, is accomplished by the interaction with the tightly bound transducer protein HtrII (halobacterial transducer) in a 2:2 complex. A transducer dimer, comprising a transmembrane domain and a cytoplasmic signaling ⁄ adaptation domain con- nected by a linker region, binds two receptor proteins within the membrane. Photo-excitation of the receptor protein (k max = 500 nm for NpSRII) induces a photocycle similar to that of bacteriorhodopsin with the photocycle intermediates labeled K, L, M, N and O. The analysis of this photocycle revealed a spectrally silent irreversible transition between two M-states (M 1 and M 2 ) with a time constant of 3 ms (6). The initial step of the photocycle is the isomerization of the retinal chromophore from all-trans to 13-cis, thereby triggering a series of conformational changes, which finally lead to activation of the bound transducer dimer. The nature and kinetics of these conformational changes were investigated by time-resolved electron paramagnetic resonance (TR-EPR) spectroscopy in combination with site-directed spin labeling (SDSL). Analysis of the data obtained with the NpSRII reconstituted in purple membrane lipids (PML) in the absence of transducer revealed a transient mobilization of the spin label side chain at residue S158R1 F (located at the outwardly oriented face of helix F) and residue L159R1 F (located at the F–G helices interface) in the cytoplasmic moiety of helix F (see Fig. 1 for position of spin labels), thus suggesting a light- induced displacement of the latter. On the other side, for helix G EPR data suggest just minor light-induced structural rearrangements with respect to the other neighboring helices. Analysis of the data obtained with the NpSRII reconstituted in PML in the presence of the transducer (2:2 complex) revealed that, in contrast to the results of the transducer-free samples, the spin label side chain S158R1 F became transiently less mobile during the photocycle (S158R1 F is located in the 2:2 complex at the F-TM2 interface). Contrarily, the increase in mobility detected for L159R1 F was still observable (7). Thus, the conformational change of helix F persists in the presence of the transducer. These observations were interpreted with an outward bending motion of the cytoplasmic half of helix F occurring during the M 1 to M 2 transition (5), and a recovery of its initial position with the reformation of the receptor ground (dark) state. This is in analogy to the motion of the corresponding helix in BR (8), which has been demonstrated by means of cryo-electron microscopy (9), X-ray structural analysis (10,11) and EPR spectroscopy (12,13). It was conclu- ded that this conformational change of helix F triggers the activation of the transducer molecule, reflected in a rotary or screw-like motion of its second transmembrane helix (TM2) as revealed by EPR spectroscopy (14) and confirmed by X-ray structure analysis (15). However, the X-ray structure of the activated state of the receptor ⁄ transducer complex revealed no †This paper is part of the Proceedings of the 12th International Conference on Retinal Proteins held at Awaji Island, Hyogo, Japan on 4–8 June 2006. *Corresponding author e-mail: hsteinho@uos.de (Heinz-Ju¨ rgen Steinhoff) Ó 2007 American Society for Photobiology 0031-8655/07 Photochemistry and Photobiology, 2007, 83: 263–272 263