Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli

Archaeal phototaxis is mediated by sensory rhodopsins which form complexes with their cognate transducers. Whereas the receptors sensory rhodopsin I and sensory rhodopsin II (SRII) have been expressed in Escherichia coli (E. coli) only shortened fragments of HtrII from Natronomonas pharaonis (NpHtrII) are available. Here we describe the heterologous expression of full length NpHtrII which was achieved in yields of up to 0.9 mg per litre cell culture. Gel filtration analysis reveals the tendency of the transducer to form dimers and higher-order oligomers which was also observed when complexed to NpSRII. A circular dichroism (CD) spectrum of NpHtrII is comparable to those obtained for the E. coli chemoreceptors indicating a similar folding with predominantly alpha-helical structure. NpHtrII dissociates from the NpSRII/HtrII complex with an apparent K(D) of about 0.6 microM. Photocycle kinetics of the complex is comparable to that obtained for NpSRII in complex with a truncated transducer with slight differences in the M-decay. The data indicate that the heterologously expressed NpHtrII adopt a native like structure, providing the means for elucidating transmembrane signal transduction and activation of microbial signalling cascades.     

Structural analysis of the phototactic transducer protein HtrII linker region from Natronomonas pharaonis

Halobacterial pharaonis phoborhodopsin [ppR, also called Natronomonas pharaonis sensory rhodopsin II (NpSRII)] is a phototaxis protein which transmits a light signal to the cytoplasm through its transducer protein (pHtrII). pHtrII, a two-transmembrane protein that interacts with ppR, belongs to the group of methyl-accepting chemotaxis proteins (MCPs). Several mutation studies have indicated that the linker region connecting the transmembrane and methylation regions is necessary for signal transduction. However, the three-dimensional (3D) structure of an MCP linker region has yet to be reported, and hence, details concerning the signal transduction mechanism remain unknown. Here the structure of the pHtrII linker region was investigated biochemically and biophysically. Following limited proteolysis, only one trypsin resistant fragment in the pHtrII linker region was identified. This fragment forms a homodimer with a Kd value of 115 microM. The 3D structure of this fragment was determined by solution NMR, and only one alpha-helix was found between two HAMP domains of the linker region. This alpha-helix was significantly stabilized within transmembrane protein pHtrII as revealed by CW-EPR. The presence of Af1503 HAMP domain-like structures in the linker region was supported by CD, NMR, and ELDOR data. The alpha-helix determined here presumably works as a mechanical joint between two HAMP domains in the linker region to transfer the photoactivated conformational change downstream.     

The cytoplasmic membrane-proximal domain of the HtrII transducer interacts with the E-F loop of photoactivated Natronomonas pharaonis sensory rhodopsin II

The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Forster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Forster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.     

Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis

The photocycle kinetics of halorhodopsin from Natronobacterium pharaonis (pHR(575)) was analyzed at different temperatures and chloride concentrations as well as various halides. Over the whole range of modified parameters the kinetics can be adequately modeled with six apparent rate constants. Assuming a model in which the observed rates are assigned to irreversible transitions of a single relaxation chain, six kinetically distinguishable states (P(1-6)) are discernible that are formed from four chromophore states (spectral archetypes S(j): K(570), L(N)(520), O(600), pHR'(575)). Whereas P(1) coincides with K(570) (S(1)), both P(2) and P(3) have identical spectra resembling L(520) (S(2)), thus representing a true spectral silent transition between them. P(4) constitutes a fast temperature-dependent equilibrium between the chromophore states S(2) and S(3) (L(520) and O(600), respectively). The subsequent equilibrium (P(5)) of the same spectral archetypes is only moderately temperature dependent but shows sensitivity toward the type of anion and the chloride concentration. Therefore, S(2) and S(3) occurring in P(4) as well as in P(5) have to be distinguished and are assigned to L(520)<--> O(1)(600) and O(2)(600)<--> N(520) equilibrium, respectively. It is proposed that P(4) and P(5) represent the anion release and uptake steps. Based on the experimental data affinities of the halide binding sites are estimated.     

Properties and photochemistry of a halorhodopsin from the haloalkalophile, Natronobacterium pharaonis

Pharaonis halorhodopsin is a light-driven transport system for chloride, similarly to the previously described halorhodopsin, but we find that it transports nitrate as effectively as chloride. We studied the photoreactions of the purified, detergent-solubilized pharaonis pigment with a gated multichannel analyzer. At a physiological salt concentration (4 M NaCl), the absorption spectra and rate constants of rise and decay for intermediates of the photocycle were similar to those for halorhodopsin. In buffer containing nitrate, halorhodopsin exhibits a second, truncated photocycle; this difference in the photoreaction of the pigment occurs when an anion is bound in such a way as to preclude transport. As expected from the lack of anion specificity in the transport, the photocycle of pharaonis halorhodopsin was nearly unaffected by replacement of chloride with nitrate. All presumed buried positively charged residues, which might play a role in anion binding, are conserved in the two pigments. At the extracellular end of the presumed helix C, however, an arginine residue is found in halorhodopsin, but not in pharaonis halorhodopsin, and an arginine-rich segment between the presumed helices A and B in halorhodopsin is replaced by a less positively charged sequence in pharaonis halorhodopsin (Lanyi, J. K., Duschl, A., Hatfield, G. W., May, K., and Oesterhelt, D. (1990) J. Biol. Chem. 265, 1253-1260). One or both of these alterations may explain the difference in the anion selectivity of the two proteins.     

Ser-130 of Natronobacterium pharaonis halorhodopsin is important for the chloride binding

Pharaonis halorhodopsin (phR) is an inward light-driven chloride ion pump from Natronobacterium pharaonis. In order to clarify the role of Ser-130(phR) residue which corresponds to Ser-115(shR) for salinarum hR on the anion-binding affinity, the wild-type and Ser-130 mutants substituted with Thr, Cys and Ala were expressed in E. coli cells and solubilized with 0.1% n-dodecyl beta-D-maltopyranoside The absorption maximum (lambda(max)) of the S130T mutant indicated a blue shift from that of the wild type in the absence and presence of chloride. For S130A, a large red shift (12 nm) in the absence of chloride was observed. The wild-type and all mutants showed the blue-shift of lambda(max) upon Cl(-) addition, from which the dissociation constants of Cl(-) were determined. The dissociation constants were 5, 89, 153 and 159 mM for the wild-type, S130A, S130T and S130C, respectively, at pH 7.0 and 25 degrees C. Circular dichroic spectra of the wild-type and the Ser-130 mutants exhibited an oligomerization. The present study revealed that the Ser-130 of N. pharaonis halorhodopsin is important for the chloride binding.     

The transducer protein HtrII modulates the lifetimes of sensory rhodopsin II photointermediates.

We studied the photochemical reaction cycle of sensory rhodopsin II (SRII) by flash photolysis of Halobacterium salinarum membranes genetically engineered to contain or to lack its transducer protein HtrII. Flash photolysis data from membranes containing HtrII were fit well in the 10 micros-10 s range by three rate constants and a linear unbranched pathway from the unphotolyzed state with 487 nm absorption maximum to a species with absorption maximum near 350 nm (M) followed by a species with maximum near 520 nm (O), as has been found in previous studies of wild-type membranes. Data from membranes devoid of HtrII exhibited similar M and O intermediates but with altered kinetics, and a third intermediate absorbing maximally near 470 nm (N) was present in an equilibrium mixture with O. The modulation of SRII photoreactions by HtrII indicates that SRII and HtrII are physically associated in a molecular complex. Arrhenius analysis shows that the largest effect of HtrII, the acceleration of O decay, is attributable to a large decrease in activation enthalpy. Based on comparison of SRII photoreactions to those of sensory rhodopsin I and bacteriorhodopsin, we interpret this kinetic effect to indicate that HtrII interacts with SRII so that it alters the reaction process involving deprotonation of Asp73, the proton acceptor from the Schiff base.     

Photocycle of phoborhodopsin from haloalkaliphilic bacterium (Natronobacterium pharaonis) studied by low-temperature spectrophotometry.

Phoborhodopsin (pR) is the fourth retinal pigment of Halobacterium halobium and works as a photoreceptor for the negative phototactic response. A similar pigment was previously found in haloalkaliphilic bacterium (Natronbacterium pharaonis) and also works as the receptor of the negative phototactic response; this pigment is called pharaonis phoborhodopsin (ppR). In this paper, the photocycle of ppR was investigated by means of low-temperature spectrophotometry. The absorption maximum of ppR is located at 498 nm, while that of pR is at 487 nm. The absorption spectra of the two have similar vibrational structures. Irradiation of ppR below -100 degrees C produced a K-like intermediate (ppRK) which was a composite of two components. The original ppR and ppRK were perfectly photoreversible. On warming, ppRK was directly converted to an M-like intermediate without formation of the L-like intermediate. The M-like intermediate was converted to the O-like intermediate at pH 7.2, but the O-like intermediate was not detected at pH 9.0. The O-like intermediate then reverted to the original pigment. On the basis of these findings, the photocycle and the primary photochemical process of ppR are presented.     

Chaperoning transmembrane helices in the lipid bilayer

Elimination of membrane proteins often requires recognition of their transmembrane domains (TMDs) in the lipid bilayer. In this issue, Arines et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202001116) show that in Saccharomyces cerevisiae, the vacuole-associated Rsp5 ubiquitin ligase uses a TMD in substrate adaptor Ssh4 to recognize membrane helices in Ypq1, which targets this lysine transporter for lysosomal degradation during lysine starvation.

In eukaryotic cells, protein quality control (PQC) mediates the degradation of not only aberrant but also unwanted polypeptides, safeguarding both the quality and quantity of the cellular proteome (1). A central goal in PQC research is to delineate the mechanism of substrate selection, which, if inappropriately executed, could lead to undesired destruction of functional proteins and thus the collapse of the proteostasis network. For soluble proteins that succumb to PQC, it is usually the surface exposure of hydrophobic elements that alerts cellular chaperones to potential folding catastrophe (2). Chaperones often serve a dual triaging role: while giving their clients additional time to fold, they can also interface with degradation machineries such as the ubiquitin proteasome system or lysosomes, causing the elimination of terminally misfolded or unwanted polypeptides.Unlike PQC of soluble proteins, substrate recognition for membrane proteins bearing abnormal transmembrane domain (TMD) is largely unknown, even for the best characterized PQC process, ER-associated degradation (ERAD; 3). Early studies on PQC of unassembled T cell receptor α chain (TCRα) showed that the single TMD of TCRα contains two charged residues, which are thermodynamically unfavored in the lipid environment and thus must be shielded when TCRα assembles with CD3σ. Accordingly, unassembled TCRα is eliminated by ERAD via a mechanism dependent on these charged residues (4), but TMD-specific chaperones responsible for recognizing charged residues in the lipid bilayer have not been identified. Likewise, recent investigations into the function of the Hrd1 ubiquitin ligase suggested a role for the TMDs of Hrd1 in recognition of specific aberrant membrane proteins in ERAD (5). Cryo-EM studies further showed two juxtaposed central cavities with a lateral gate poised to receive TMDs in the yeast Hrd1 complex (6), but how aberrant TMDs in ERAD substrates are recognized and retrotranslocated by Hrd1 remains an open question.The issue of substrate recognition becomes even more complex for feedback-regulated degradation of unwanted membrane proteins. In this case, substrates are initially stable and functionally essential, but a change in environmental cues renders them dispensable and results in a short-lived fate. One such example is the sterol-regulated degradation of a sterol-synthesizing enzyme called HMG-CoA reductase (HMGR). HMGR is a stable ER protein when the sterol level is low, but an increase in membrane sterol abundance alters the conformation of a sterol-sensing domain in HMGR, exposing an element functionally equivalent to a degron in short-lived proteasomal substrates (7). Despite extensive studies, the molecular signature of the degron in HMGR is still undefined, let alone the molecular basis of its recognition. In this issue, Arines and colleagues investigate how Ypq1, a multi-spanning lysine transporter of the yeast vacuole, is regulated by lysine availability, a regulated membrane protein turnover event analogous to HMGR degradation. Their study identifies critical residues in Ypq1 TMDs for its turnover and establishes the Rsp5 ubiquitin ligase adaptor Ssh4 as a TMD-specific chaperone that recognizes these elements (8).Ypq1 is a seven-transmembrane, PQ loop–containing lysine transporter localized to the yeast vacuole membrane. Under lysine-replete conditions, Ypq1 is stable as it uses a PQ loop–dependent conformational cycle to import excess lysine into the vacuole. When lysine is depleted, Ypq1 is sorted into the multivesicular body (MVB) for degradation (Fig. 1). This process is initiated once Ypq1 is ubiquitinated by the ubiquitin ligase complex Rsp5–Ssh4, but how Ypq1 is targeted by Rsp5–Ssh4 has been unclear (9).Open in a separate windowFigure 1.Regulated recognition of Ypq1 by Ssh4. When lysine in the cytosol is abundant, Ypq1 undergoes a rapid conformational cycle to transport lysine from the cytosol into the vacuole lumen. Under lysine-depleted conditions, the transporter is trapped in a conformation recognizable by Ssh4, which recruits Rsp5 to catalyze Ypq1 ubiquitination and internalization into the MVB. Ub, ubiquitin.To understand the mechanism of Ypq1 recognition, Arines et al. first engineered a Ypq1 mutant that uncouples ligase-mediated degradation from lysine availability. This Ypq1 mutant is constitutively degraded in an Ssh4-dependent manner even under lysine-replete conditions. With this tool in hand, they performed a random mutagenesis-based suppressor screen, which identified many suppressor mutants. Mapping these mutations revealed several elements in Ypq1 that are critical for ligase recognition, which include two TMDs (TM5 and TM7) and a cytosolic loop. Importantly, when these mutations were introduced back into wild-type Ypq1, they also block Ssh4-dependent, lysine-regulated Ypq1 degradation. As expected, coimmunoprecipitation showed that Ypq1 suppressor mutants have reduced affinity to Ssh4. Since the cytosolic loop contains a previously known Rsp5 recognition motif, they further characterized the role of Ypq1 TMDs in ligase recruitment.Structural modeling suggests that TM5 and TM7 are juxtaposed to each other. Systematic mutagenesis targeting each residue of these two TMDs further consolidated the residues essential for Ssh4-mediated degradation. A similar mutagenesis study on Ssh4 revealed an important role for the Ssh4 TMD in Ypq1 degradation. Interestingly, for both Ssh4 and Ypq1, many identified residues are clustered on one side of the membrane helices. Arines et al. propose that Ssh4 uses its TMD to recognize TM5 and TM7 in Ypq1 based on a charge complementation experiment: a charged residue introduced into TM5 of Ypq1 abolished Ssh4-mediated degradation, but introducing an opposite charge into the TMD of Ssh4 restored lysine-regulated Ypq1 degradation.The recognition of Ypq1 by Ssh4 appears to occur when Ypq1 adopts a specific conformation during lysine transport because charge complementarity–based degradation of Ypq1 depends on the PQ loop, which is required for lysine transport. Additionally, structural modeling of Ypq1 suggested that in the inward-open and occluded conformations, TM5 is packed against TM7, but the two TMDs become distant from each other in the outward-open conformation, which exposes residues critical for Ssh4 recognition. These findings suggest that the rapid conformational cycling during lysine transport may prevent Ssh4 recognition, but lysine depletion stalls Ypq1 in a conformation recognizable by Ssh4 (Fig. 1).The study, together with the recent discovery of the ER membrane protein complex (EMC) in the biogenesis of multi-spanning membrane proteins at the ER, suggests a new class of chaperones that recognize specific features in TMDs. While emerging evidence suggests that the EMC recognizes exposed charged or polar residues in TMDs (10), the molecular basis of Ssh4 substrate interaction remains unclear. Like cytosolic chaperones, the EMC at the ER appears to play a dual role: while initially shielding charged/polar residues to facilitate TMD assembly, it may eventually target misassembled membrane proteins for degradation. By contrast, TMD-specific chaperones in other organelles like Ssh4 may have a more dedicated function in PQC. Clearly, more TMD-specific chaperones await to be discovered. Additionally, future studies will surely reveal not only the range of substrates and functions for each TMD-specific chaperone but also the structural basis of TMD recognition.     

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.     

Light-induced structural changes during incubation of isolated maize etioplasts

Summary Etioplasts were isolated from leaves of 9-day-old etiolated maize (Zea mays L.) seedlings and incubated in a relatively simple medium in light and in the dark. During the first 5 h no changes occurred in the fine structure of the isolated etioplasts in the dark. In light the size of the prolamellar bodies decreased and significantly more plastid sections without prolamellar bodies were counted. The total length of the thylakoids per plastid section increased, but there was no evidence for bi- and polythylakoid formation. It is concluded that light induces the structural transformation of the prolamellar body membranes into primary thylakoids also in isolated etioplasts.     

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.     

The photophobic receptor from Natronobacterium pharaonis: temperature and pH dependencies of the photocycle of sensory rhodopsin II.

The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.     

Structural changes of the complex between pharaonis phoborhodopsin and its cognate transducer upon formation of the M photointermediate

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes, and the association is weakened by 2 orders of magnitude in the M intermediate. Such change is believed to correspond to the transfer of the light signal to pHtrII. In this paper, we applied Fourier transform infrared (FTIR) spectroscopy to the active M intermediate in the absence and presence of pHtrII. The obtained difference FTIR spectra were surprisingly similar, notwithstanding the presence of pHtrII. This result strongly suggests that the transducer activation in the ppR-pHtrII system does not induce secondary structure alterations of the pHtrII itself. On the other hand, we found that the hydrogen bond of the OH group of Thr204 is altered in the primary K intermediate, but restored in the M intermediate. The hydrogen bond of Asn74 in pHtrII is strengthened in M, presumably because of the change in interaction with Tyr199 of ppR. These facts provided a light signaling pathway from Lys205 (retinal) of the receptor to Asn74 of the transducer through Thr204 and Tyr199. Transducer activation is likely to involve a relaxation of Thr204 in the receptor and hydrogen bonding alteration of Asn74 in the transducer, during which the helices of the transducer perform rigid-body motion without changing their secondary structures.     

Light-induced global structural changes in phytochrome A regulating photomorphogenesis in plants

Phytochromes are photoreceptor proteins that monitor the light environment and regulate a variety of photomorphogenic responses to optimize the growth and development of plants. Phytochromes comprise N-terminal photosensory and C-terminal regulatory domains. They are mutually photoconvertible between a red-light-absorbing (Pr) and a far-red-light-absorbing (Pfr) form. Their interconversion by light stimuli initiates downstream signaling cascades. Here we report the molecular structures of pea phytochrome A lacking the N-terminal 52 amino-acid residues in the Pr and Pfr forms studied by small-angle X-ray scattering. A new purification protocol yielded monodispersive sample solutions. The molecular mass and the maximum dimension of Pr determined from scattering data indicated its dimeric association. The molecular structure of Pr predicted by applying the ab initio simulation method to the scattering profile was approximated as a stack of two flat bodies, comprising two lobes assignable to the functional regions. Scattering profiles recorded under red-light irradiation showed small but definite changes from those of Pr. The molecular dimensions and predicted molecular structure of Pfr suggest global structural changes such as movement of the C-terminal domains in the Pr-to-Pfr phototransformation. Red-light-induced structural changes in Pfr were reversible, mostly due to thermal relaxation processes.     

Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers

Amphipathic helices in membrane proteins that interact with the hydrophobic/hydrophilic interface of the lipid bilayer have been difficult to structurally characterize. Here, the backbone structure and orientation of an amphipathic helix in the full-length M2 protein from influenza A virus has been characterized. The protein has been studied in hydrated DMPC/DMPG lipid bilayers above the gel to liquid-crystalline phase transition temperature by solid-state NMR spectroscopy. Characteristic PISA (Polar Index Slant Angle) wheels reflecting helical wheels have been observed in uniformly aligned bilayer preparations of both uniformly 15N labeled and amino acid specific labeled M2 samples. Hydrogen/deuterium exchange studies have shown the very slow exchange of some residues in the amphipathic helix and more rapid exchange for the transmembrane helix. These latter results clearly suggest the presence of an aqueous pore. A variation in exchange rate about the transmembrane helical axis provides additional support for this claim and suggests that motions occur about the helical axes in this tetramer to expose the entire backbone to the pore.     

Signal transmission through the HtrII transducer alters the interaction of two alpha-helices in the HAMP domain

A conformational change of the transducer HtrII upon photoexcitation of the associated photoreceptor sensory rhodopsin II (SRII) was investigated by monitoring the kinetics of volume changes and the diffusion coefficient (D) of the complex during the photochemical reaction cycle. To localize the region of the transducer responsible, we truncated it at various positions in the cytoplasmic HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain. The truncations do not alter receptor binding, which is dependent primarily on membrane-embedded domain interactions. We found that the light-induced reduction in D occurs in transducers of lengths 120 and 157 residues (Tr120 and Tr157), which are both predicted to contain a HAMP domain consisting of two amphipathic α-helices (AS-1 and AS-2). In contrast, the change in D was abolished in a transducer of 114 amino acid residues (Tr114), which lacks a distal portion of the second α-helix AS-2. The volume changes in SRII-Tr114 are comparable in amplitude and kinetics with those in SRII-Tr120 and SRII-Tr157, confirming the integrity of the complex, which was previously concluded from the similar SRII binding affinity and similar blocking of SRII proton transport by full-length HtrII and Tr114. Our results indicate that a substantial conformational change occurs in the HAMP domain during SRII-HtrII signaling. The data presented here are the first demonstration of stimulus-induced conformational changes of a HAMP domain and provide evidence that the presence of AS-2 is crucial for the conformational alterations. The reduction in diffusion coefficient is likely to due to structural changes in the AS-1 and AS-2 helices such that hydrogen bonding with the surrounding water molecules is increased, thereby increasing friction with the solvent. Similar structural changes may be a general feature in HAMP domain switching, which occurs in diverse signaling proteins, including sensor kinases, taxis receptors/transducers, adenylyl cyclases, and phosphatases.     

Light-induced rotation of a transmembrane alpha-helix in bacteriorhodopsin

Spin labeling EPR spectroscopy has been used to characterize light-induced conformational changes of bacteriorhodopsin (bR). Pairs of nitroxide spin labels were attached to engineered cysteine residues at strategic positions near the cytoplasmic ends of transmembrane alpha-helices B, F, and G in order to monitor distance changes upon light activation. The EPR analysis of six doubly labeled bR mutants indicates that the cytoplasmic end of helix F not only tilts outwards, but also rotates counter-clockwise during the photocycle. The direction of the rotation of helix F is the opposite of the clockwise rotation previously reported for bovine rhodopsin. The opposite chirality of the F helix rotation in the two systems is perhaps related to the differences in the cis-trans photoisomerization of the retinal in the two proteins. Using time-resolved EPR, we monitored the rotation of helix F also in real time, and found that the signal from the rotation arises concurrently with the reprotonation of the retinal Schiff base.