Molecular Impact of the Membrane Potential on the Regulatory Mechanism of Proton Transfer in Sensory Rhodopsin II

Metabolism establishes a potential difference across the cell membrane of every living cell which drives and regulates secondary ion and solute transfer across membrane proteins. Unraveling the effect of the membrane potential on the level of single molecular groups of the membrane protein was long hampered by the lack of appropriate analytical techniques. We have developed Surface Enhanced Infrared Difference Absorption Spectroscopy (SEIDAS), a highly sensitive vibrational technique for surface analysis, for the study of solid-supported monolayers of orientated membrane proteins. Here, we present spectroscopic data on vibrational changes of sensory rhodopsin II from Natronomonas pharaonis (NpSR II). The application of the electrode potential provides a voltage drop across the NpSR II monolayer through the Helmholtz double layer that mimics the cellular membrane potential. IR difference spectra indicated a shift of the photostationary equilibrium from an M and O mixture toward an M dominant equilibrium. The shift of positive to negative potential exhibited similar effects on the light-induced SEIDA spectra as the increase in pH. This effect is explained in terms of local pH change raised by the compensation of excess charge from the electrode. As we have shown earlier (Jiang, et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105 (34), 12113−12117), the application of an electric field opposite to the physiological proton transfer from the retinal Schiff base to its counterion Asp75 leads to the selective halt of the latter. However, when the solution pH is much higher than 5.8, that is, when the proton releasing group at the extracellular side is ionized, proton transfer of Asp75 becomes insensitive to the electric field exerted by the electrode. We infer that the deprotonation of the proton release group creates a local polar environment surrounding Asp75 as a consequence of hydrogen-bonding rearrangements that exceeds the energy of the external dipole. Our results reveal a molecular model for the physiological regulation of the photocycle of NpSR II by the potential drop across the membrane which came about by the interplay between the change in local pH at the membrane surface and the external electric field

Complexation of Metals with Piperazine-Containing Azamacrocyclic Fluorophores

Azamacrocyclic fluorophores containing piperazine units were synthesized using sequential rhodium-catalyzed regioselective hydroformylation−reductive amination. A piperazine unit is introduced into the macrocycles to act simultaneously as electron donor and binding site. The macrocycles chelate divalent cations, either Zn2+ or Co2+, which considerably enhanced fluorescence. Complexation with Zn2+ was additionally confirmed by NMR

Transient Conformational Changes of Sensory Rhodopsin II Investigated by Vibrational Stark Effect Probes

Sensory rhodopsin II (SRII) is the primary light sensor in the photophobic reaction of the halobacterium Natronomonas pharaonis. Photoactivation of SRII results in a movement of helices F and G of this seven-helical transmembrane protein. This conformational change is conveyed to the transducer protein (HtrII). Global changes in the protein backbone have been monitored by IR difference spectroscopy by recording frequency shifts in the amide bands. Here we investigate local structural changes by judiciously inserting thiocyanides at different locations of SRII. These vibrational Stark probes absorb in a frequency range devoid of any protein vibrations and respond to local changes in the dielectric, electrostatics, and hydrogen bonding. As a proof of principle, we demonstrate the use of Stark probes to test the conformational changes occurring in SRII 12 ms after photoexcitation and later. Thus, a methodology is provided to trace local conformational changes in membrane proteins by a minimal invasive probe at the high temporal resolution inherent to IR spectroscopy

Intra-dimer distance changes between related residues of the transducer upon demethylation

<p>A: Structure of the <i>Np</i>SRII/<i>Np</i>HtrII trimeric complex with distance changes color coded (calculated as an average over the three dimers). Positive values of the distance difference (blue) indicate a looser packing of the corresponding residues in the demethylated system, negative values (red) indicate a more compact packing. B: The intra-dimer distance difference as function of residue number shows distinct changes in the transmembrane region of the complex, an inversion of the packing densities for the two HAMP domains, minor changes at the methylation sites (m.s.), and a decrease of the packing density in the CheA/CheW binding site region labeled A/W.</p

List of simulations performed.

<p>List of simulations performed.</p

Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of <i>Natronomonas pharaonis</i>

<div><p>Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely <i>Np</i>SRII/<i>Np</i>HtrII from <i>Natronomonas pharaonis</i>. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of <i>Np</i>SRII/<i>Np</i>HtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for <i>E</i>. <i>coli</i> chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.</p></div

Two component phototaxis system of <i>N</i>. <i>pharaonis</i>.

<p><i>Np</i>SRII/<i>Np</i>HtrII dimers are the basic elements of photoreceptor complexes in <i>N</i>. <i>pharaonis</i>. They consist of two sensory rhodopsins, <i>Np</i>SRII, and two transducer proteins, <i>Np</i>HtrII, mostly of α-helical secondary structure, with a characteristic domain organization. Light activation of <i>Np</i>SRII induces conformational and/or dynamical changes in the transducer which are converted by two HAMP domains and conveyed along the 20 nm long transducer to the tip region, where it activates the homodimeric histidine kinase CheA bound together with the adapter protein CheW. The kinase CheA undergoes auto-phosphorylation and further transfers the phosphate group to the response regulators CheY or CheB. CheY affects the rotational bias of the flagellar motor, while the methylesterase CheB along with the methyltransferase CheR controls the adaptation (feedback) mechanism. The related chemoreceptor and most likely also the photoreceptor dimers further organize into trimers, which, together with CheA and CheW, lead to the formation of large sensor arrays.</p

Inter-dimeric distances for related residues of the transducer.

<p>Distances were calculated as an average over the three dimers for the methylated (black) and demethylated (red) states, shaded areas representing the standard deviation. The distance is measured between the center of mass (COM) of two related residues in one dimer and the COM of the six respective residues in the trimer-of-dimers (see inset on the lower left). The domains of the complex are depicted in colored bars; m.s and A/W indicate methylation sites and binding sites for CheA/CheW, respectively. Representative distance trajectories are depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004561#pcbi.1004561.s007" target="_blank">S7 Fig</a>.</p

Model for the <i>Np</i>SRII/<i>Np</i>HtrII complex activation.

<p>The regions with higher mobility are shown in diffuse representation; the arrows correspond to the domain motions (compacting/expanding within the trimer).</p

Conformations of the trimeric photoreceptor-transducer complexes.

<p>Cartoon with the resulting structures of the demethylated (left) and the methylated (right) trimer systems combined in the bent membrane with a schematic representation of the adaptation process. Methylation sites are shown as red and black spheres in the demethylated and methylated state, respectively.</p