Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsin's retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.

Membrane proteins are molecular machines that transport ions, solutes, or information across the cell membrane. Electrophysiological techniques have unraveled many functional aspects of ion channels but suffer from the lack of structural sensitivity. Here, we present spectroelectrochemical data on vibrational changes of membrane proteins derived from a single monolayer. ion transfer ͉ membrane potential ͉ proton translocation ͉ vibrational spectroscopy ͉ sensory rhodopsin

Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.

Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg135 3.50 and Met257 6.40 (1)] on the intradiscal (or extracellular) side of the receptor. Absorption of light drives the 11-cis-to trans-isomerization of the retinal within a tight binding pocket. The conformational changes that occur in this process must be transmitted through the membrane-spanning portion of the bilayer to the intracellular surface in order to open up the binding site for the heterotrimeric G protein, transducin. The crystal structure of the dark, inactive state of the visual pigment rhodopsin (2) reveals a tightly packed bundle of seven transmembrane (TM) helices but offers few clues as to how the helices move upon light activation.Site-directed spin-labeling studies by Hubbell and coworkers (3,4) showed that the largest change in the seven-TM-helix bundle involves an outward rotation of helix H6, consistent with an increase in volume of the receptor upon activation (5). The challenge for obtaining a high-resolution structure of the active metarhodopsin II (Meta II) intermediate has been that light activation causes the dark-state crystals of rhodopsin to dissolve (6), suggesting that the structural changes are sufficiently large to disrupt crystal packing. Salom et al. (7) were able to determine the crystal structure to 4.15-Å resolution of a photointermediate of rhodopsin containing retinal with a deprotonated Schiff base (SB) (7). The structure did not exhibit the large helix motions characteristic of the activated receptor, suggesting that this intermediate corresponds to the Meta II substate (Meta IIa) formed prior to helix motion (8).More recently, Park et al. determined the structure of opsin (9). Opsin is formed when the Meta II intermediate decays and releases the agonist all-trans-retinal from the retinal-binding site. Opsin has low (≤1%), but detectable, basal activity in rod outer segment cell membranes (10). At pH 4, FTIR difference spectra of opsin exhibit vibrational bands characteristic of Meta II (11), suggesting that opsin adopts an active conformation. The crystals of opsin obtained at pH 6 appear to retain many features characteristic of the active state (Fig. 1). In fact, the most recent crystal structure of opsin (12) contains the bound C-terminal peptide of the Gα subunit of transducin in a conformation similar to that observed in solution NMR studies on the activated Meta II intermediate (13,14).One of the most striking features of the opsin structure is that the ionic lock involving Glu134 3.49 -Arg135 3.50 of the conserved ERY sequence on H3 and Glu247 6.30 on H6 is disrupted (Fig. 1B). The opsin structur...

Photon absorption by rhodopsin is proposed to lead to an activation pathway that is described by the extended reaction scheme: Meta I ⇋ Meta II a ⇋ Meta II b ⇋ Meta II b H + ; where Meta II b H + is thought to be the conformational substate that activates the G protein transducin. Here we test this extended scheme for rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activation mechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), selectively stabilize the Meta II a substate in the above mechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structural and functional changes involved in the transition to the Meta II a intermediate, which links the inactive Meta I intermediate with the Meta II b states formed by helix re-arrangement. Besides the opening of the Schiff base ionic lock, the Meta II a substate is characterized by an activation switch in a conserved water-mediated hydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residue Asp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3/H5 network mediated by His211 and Glu122 is absent in Meta II a . The latter rearrangement takes place only in the subsequent transition to Meta II b , which has been previously associated with movement of H6. Our results imply that activating structural changes in the H1/ H2/H7 network are triggered by disruption of the Schiff base salt bridge, and occur prior to other chromophore-induced changes in the H3/H5 network and the outward tilt of H6 in the activation process.