Biomedical subjects

John L Spudich

Publications and source records attributed to John L Spudich.

At least 19 recordsLinked to original sources

PubMed2006

Laser-induced transient grating analysis of dynamics of interaction between sensory rhodopsin II D75N and the HtrII transducer.

The interaction between sensory rhodopsin II (SRII) and its transducer HtrII was studied by the time-resolved laser-induced transient grating method using the D75N mutant of SRII, which exhibits minimal visible light absorption changes during its photocycle, but mediates normal phototaxis responses. Flash-induced transient absorption spectra of transducer-free D75N and D75N joined to 120 amino-acid residues of the N-terminal part of the SRII transducer protein HtrII (DeltaHtrII) showed only one spectrally distinct K-like intermediate in their photocycles, but the transient grating method resolved four intermediates (K(1)-K(4)) distinct in their volumes. D75N bound to HtrII exhibited one additional slower kinetic species, which persists after complete recovery of the initial state as assessed by absorption changes in the UV-visible region. The kinetics indicate a conformationally changed form of the transducer portion (designated Tr*), which persists after the photoreceptor returns to the unphotolyzed state. The largest conformational change in the DeltaHtrII portion was found to cause a DeltaHtrII-dependent increase in volume rising in 8 micros in the K(4) state and a drastic decrease in the diffusion coefficient (D) of K(4) relatively to those of the unphotolyzed state and Tr*. The magnitude of the decrease in D indicates a large structural change, presumably in the solvent-exposed HAMP domain of DeltaHtrII, where rearrangement of interacting molecules in the solvent would substantially change friction between the protein and the solvent.

Archaeal Proteins↗

PubMed2006

Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor.

In haloarchaea, light-driven ion transporters have been modified by evolution to produce sensory receptors that relay light signals to transducer proteins controlling motility behavior. The proton pump bacteriorhodopsin and the phototaxis receptor sensory rhodopsin II (SRII) differ by 74% of their residues, with nearly all conserved residues within the photoreactive retinal-binding pocket in the membrane-embedded center of the proteins. Here, we show that three residues in bacteriorhodopsin replaced by the corresponding residues in SRII enable bacteriorhodopsin to efficiently relay the retinal photoisomerization signal to the SRII integral membrane transducer (HtrII) and induce robust phototaxis responses. A single replacement (Ala-215-Thr), bridging the retinal and the membrane-embedded surface, confers weak phototaxis signaling activity, and the additional two (surface substitutions Pro-200-Thr and Val-210-Tyr), expected to align bacteriorhodopsin and HtrII in similar juxtaposition as SRII and HtrII, greatly enhance the signaling. In SRII, the three residues form a chain of hydrogen bonds from the retinal's photoisomerized C(13)=C(14) double bond to residues in the membrane-embedded alpha-helices of HtrII. The results suggest a chemical mechanism for signaling that entails initial storage of energy of photoisomerization in SRII's hydrogen bond between Tyr-174, which is in contact with the retinal, and Thr-204, which borders residues on the SRII surface in contact with HtrII, followed by transfer of this chemical energy to drive structural transitions in the transducer helices. The results demonstrate that evolution accomplished an elegant but simple conversion: The essential differences between transport and signaling proteins in the rhodopsin family are far less than previously imagined.

Bacteriorhodopsins↗

PubMed2006

Role of the cytoplasmic domain in Anabaena sensory rhodopsin photocycling: vectoriality of Schiff base deprotonation.

Light-induced electric signals in intact E. coli cells generated by heterologously expressed full-length and C-terminally truncated versions of Anabaena sensory rhodopsin (ASR) demonstrate that the charge movements within the membrane-embedded part of the molecule are stringently controlled by the cytoplasmic domain. In particular, truncation inverts the direction of proton movement during Schiff base deprotonation from outward to cytoplasmic. Truncation also alters faster charge movements that occur before Schiff base deprotonation. Asp(217) as previously shown by FTIR serves as a proton acceptor in the truncated ASR but not in the full-length version, and its mutation to Asn restores the natural outward direction of proton movement. Introduction of a potential negative charge (Ser(86) to Asp) on the cytoplasmic side favors a cytoplasmic direction of proton release from the Schiff base. In contrast, mutation of the counterion Asp(75) to Glu reverses the photocurrent to the outward direction in the truncated pigment, and in both truncated and full-length versions accelerates Schiff base deprotonation more than 10-fold. The communication between the cytoplasmic domain and the membrane-embedded photoactive site of ASR demonstrated here is likely to derive from the receptor's use of a cytoplasmic protein for signal transduction, as has been suggested previously from binding studies.

Anabaena↗

PubMed2006

Microbial rhodopsins: functional versatility and genetic mobility.

The type 1 (microbial) rhodopsins are a diverse group of photochemically reactive proteins that span the three domains of life. Their broad phylogenetic distribution has motivated conjecture that rhodopsin-like functionality was present in the last common ancestor of all life. Here, we discuss the evolution of the type 1 microbial rhodopsins and document five cases of lateral gene transfer (LGT) between domains. We suggest that, thanks to the functional versatility of these retinylidene proteins and the relative ease with which they can complement the existing energy-generating or photosensory repertoires of many organisms, LGT is in fact the principal force that determines their broad but patchy distribution.

Evolution, Molecular↗

PubMed2006

The multitalented microbial sensory rhodopsins.

Sensory rhodopsins are photoactive, membrane-embedded seven-transmembrane helix receptors that use retinal as a chromophore. They are widespread in the microbial world in each of the three domains of life: Archaea, Bacteria and Eukarya. A striking characteristic of these photoreceptors is their different modes of signaling in different organisms, including interaction with other membrane proteins, interaction with cytoplasmic transducers and light-controlled Ca(2+) channel activity. More than two decades since the discovery of the first sensory rhodopsins in the archaeon Halobacterium salinarum, genome projects have revealed a widespread presence of homologous photosensors. New work on cyanobacteria, algae, fungi and marine proteobacteria is revealing how evolution has modified the common design of these proteins to produce a remarkably rich diversity in their signaling biochemistry.

Models, Biological↗

PubMed2006

Functional importance of the interhelical hydrogen bond between Thr204 and Tyr174 of sensory rhodopsin II and its alteration during the signaling process.

Sensory rhodopsin II (SRII), a receptor for negative phototaxis in haloarchaea, transmits light signals through changes in protein-protein interaction with its transducer HtrII. Light-induced structural changes throughout the SRII-HtrII interface, which spans the periplasmic region, membrane-embedded domains, and cytoplasmic domains near the membrane, have been identified by several studies. Here we demonstrate by site-specific mutagenesis and analysis of phototaxis behavior that two residues in SRII near the membrane-embedded interface (Tyr174 on helix F and Thr204 on helix G) are essential for signaling by the SRII-HtrII complex. These residues, which are the first in SRII shown to be required for phototaxis function, provide biological significance to the previous observation that the hydrogen bond between them is strengthened upon the formation of the earliest SRII photointermediate (SRII(K)) only when SRII is complexed with HtrII. Here we report frequency changes of the S-H stretch of a cysteine substituted for SRII Thr204 in the signaling state intermediates of the SRII photocycle, as well as an influence of HtrII on the hydrogen bond strength, supporting a direct role of the hydrogen bond in SRII-HtrII signal relay chemistry. Our results suggest that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174, which is located at both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface to the signal-bearing second transmembrane helix of HtrII. The results argue for a critical process in signal relay occurring at this membrane interfacial region of the complex.

Cell Membrane↗

PubMed2006

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal.

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.

Anabaena↗

PubMed2005

Rhodopsin-mediated photoreception in cryptophyte flagellates.

We show that phototaxis in cryptophytes is likely mediated by a two-rhodopsin-based photosensory mechanism similar to that recently demonstrated in the green alga Chlamydomonas reinhardtii, and for the first time, to our knowledge, report spectroscopic and charge movement properties of cryptophyte algal rhodopsins. The marine cryptophyte Guillardia theta exhibits positive phototaxis with maximum sensitivity at 450 nm and a secondary band above 500 nm. Variability of the relative sensitivities at these wavelengths and light-dependent inhibition of phototaxis in both bands by hydroxylamine suggest the involvement of two rhodopsin photoreceptors. In the related freshwater cryptophyte Cryptomonas sp. two photoreceptor currents similar to those mediated by the two sensory rhodopsins in green algae were recorded. Two cDNA sequences from G. theta and one from Cryptomonas encoding proteins homologous to type 1 opsins were identified. The photochemical reaction cycle of one Escherichia-coli-expressed rhodopsin from G. theta (GtR1) involves K-, M-, and O-like intermediates with relatively slow (approximately 80 ms) turnover time. GtR1 shows lack of light-driven proton pumping activity in E. coli cells, although carboxylated residues are at the positions of the Schiff base proton acceptor and donor as in proton pumping rhodopsins. The absorption spectrum, corresponding to the long-wavelength band of phototaxis sensitivity, makes this pigment a candidate for one of the G. theta sensory rhodopsins. A second rhodopsin from G. theta (GtR2) and the one from Cryptomonas have noncarboxylated residues at the donor position as in known sensory rhodopsins.

Amino Acid Sequence↗

PubMed2005

New insights into metabolic properties of marine bacteria encoding proteorhodopsins.

Proteorhodopsin phototrophy was recently discovered in oceanic surface waters. In an effort to characterize uncultured proteorhodopsin-exploiting bacteria, large-insert bacterial artificial chromosome (BAC) libraries from the Mediterranean Sea and Red Sea were analyzed. Fifty-five BACs carried diverse proteorhodopsin genes, and we confirmed the function of five. We calculate that proteorhodopsin-exploiting bacteria account for 13% of microorganisms in the photic zone. We further show that some proteorhodopsin-containing bacteria possess a retinal biosynthetic pathway and a reverse sulfite reductase operon, employed by prokaryotes oxidizing sulfur compounds. Thus, these novel phototrophs are an unexpectedly large and metabolically diverse component of the marine microbial surface water.

Bacterial Proteins↗

PubMed2005

Photoactivation perturbs the membrane-embedded contacts between sensory rhodopsin II and its transducer.

The photoactivation mechanism of the sensory rho-dopsin II (SRII)-HtrII receptor-transducer complex of Natronomonas pharaonis was investigated by time-resolved Fourier transform infrared difference spectroscopy to identify structural changes associated with early events in the signal relay mechanism from the receptor to the transducer. Several prominent bands in the wild-type SRII-HtrII spectra are affected by amino acid substitutions at the receptor Tyr(199) and transducer Asn(74) residues, which form a hydrogen bond between the two proteins near the middle of the bilayer. Our results indicate disappearance of this hydrogen bond in the M and O photointermediates, the likely signaling states of the complex. This event represents one of the largest light-induced alterations in the binding contacts between the receptor and transducer. The vibrational frequency changes suggest that Asn(74) and Tyr(199) form other stronger hydrogen bonds in the M state. The light-induced disruption of the Tyr(199)-Asn(74) bond also occurs when the Schiff base counterion Asp(75) is replaced with a neutral asparagine. We compared the decrease in intensity of difference bands assigned to the Tyr(199)-Asn(74) pair and to chromophore and protein groups of the receptor at various time points during the recovery of the initial state. All difference bands exhibit similar decay kinetics indicating that reformation of the Tyr(199)-Asn(74) hydrogen bond occurs concomitantly with the decay of the M and O photointermediates. This work demonstrates that the signal relay from SRII to HtrII involves early structural alterations in the deeply membrane-embedded domain of the complex and provides a spectroscopic signal useful for correlation with the downstream events in signal transduction.

Amino Acid Substitution↗

PubMed2005

Photochromicity of Anabaena sensory rhodopsin, an atypical microbial receptor with a cis-retinal light-adapted form.

We characterize changes in isomeric states of the retinylidene chromophore during light-dark adaptation and photochemical reactions of Anabaena (Nostoc) sp. PCC7120 sensory rhodopsin (ASR). The results show that ASR represents a new type of microbial rhodopsin with a number of unusual characteristics. The three most striking are: (i) a primarily all-trans configuration of retinal in the dark-adapted state and (ii) a primarily 13-cis light-adapted state with a blue-shifted and lower extinction absorption spectrum, opposite of the case of bacteriorhodopsin; and (iii) efficient reversible light-induced interconversion between the 13-cis and all-trans unphotolyzed states of the pigment. The relative amount of ASR with cis and trans chromophore forms depends on the wavelength of illumination, providing a mechanism for single-pigment color sensing analogous to that of phytochrome pigments. In addition ASR exhibits unusually slow formation of L-like and M-like intermediates, with a dominant accumulation of M during the photocycle. Co-expression of ASR with its putative cytoplasmic transducer protein shifts the absorption maximum and strongly decreases the rate of dark adaptation of ASR, confirming interaction between the two proteins. Thus ASR, the first non-haloarchaeal sensory rhodopsin characterized, demonstrates the diversity of photochemistry of microbial rhodopsins. Its photochromic properties and the position of its two ground state absorption maxima suggest it as a candidate for controlling differential photosynthetic light-harvesting pigment synthesis (chromatic adaptation) or other color-sensitive physiological responses in Anabaena cells.

Anabaena↗

PubMed2004

Anabaena sensory rhodopsin: a photochromic color sensor at 2.0 A.

Microbial sensory rhodopsins are a family of membrane-embedded photoreceptors in prokaryotic and eukaryotic organisms. Structures of archaeal rhodopsins, which function as light-driven ion pumps or photosensors, have been reported. We present the structure of a eubacterial rhodopsin, which differs from those of previously characterized archaeal rhodopsins in its chromophore and cytoplasmic-side portions. Anabaena sensory rhodopsin exhibits light-induced interconversion between stable 13-cis and all-trans states of the retinylidene protein. The ratio of its cis and trans chromophore forms depends on the wavelength of illumination, thus providing a mechanism for a single protein to signal the color of light, for example, to regulate color-sensitive processes such as chromatic adaptation in photosynthesis. Its cytoplasmic half channel, highly hydrophobic in the archaeal rhodopsins, contains numerous hydrophilic residues networked by water molecules, providing a connection from the photoactive site to the cytoplasmic surface believed to interact with the receptor's soluble 14-kilodalton transducer.

Anabaena↗

PubMed2004

Structural changes in the photoactive site of proteorhodopsin during the primary photoreaction.

Proteorhodopsin (PR), found in marine gamma-proteobacteria, is a newly discovered light-driven proton pump similar to bacteriorhodopsin (BR). Because of the widespread distribution of proteobacteria in the worldwide oceanic waters, this pigment may contribute significantly to the global solar energy input in the biosphere. We examined structural changes that occur during the primary photoreaction (PR --> K) of wild-type pigment and two mutants using low-temperature FTIR difference spectroscopy. Several vibrations detected in the 3500-3700 cm(-1) region are assigned on the basis of H(2)O --> H(2)(18)O exchange to the perturbation of one or more internal water molecules. Substitution of the negatively charged Schiff base counterion, Asp97, with the neutral asparagine caused a downshift of the ethylenic (C=C) and Schiff base (C=N) stretching modes, in agreement with the 27 nm red shift of the visible lambda(max). However, this replacement did not alter the normal all-trans to 13-cis isomerization of the chromophore or the environment of the detected water molecule(s). In contrast, substitution of Asn230, which is in a position to interact with the Schiff base, with Ala induces a 5 nm red shift of the visible lambda(max) and alters the PR chromophore structure, its isomerization to K, and the environment of the detected internal water molecules. The combination of FTIR and site-directed mutagenesis establishes that both Asp97 and Asn230 are perturbed during the primary phototransition. The environment of Asn230 is further altered during the thermal decay of K. These results suggest that significant differences exist in the conformational changes which occur in the photoactive sites of proteorhodopsin and bacteriorhodopsin during the primary photoreaction.

Amino Acid Substitution↗

PubMed2004

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.

Archaeal Proteins↗

PubMed2004

Five residues in the HtrI transducer membrane-proximal domain close the cytoplasmic proton-conducting channel of sensory rhodopsin I.

Transducer-free sensory rhodopsins carry out light-driven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous NpHtrII to interact with the E-F loop of its cognate receptor NpSRII in the accompanying article (Yang, C.-S., Sineshchekov, O., Spudich, E. N., and Spudich, J. L. (2004) J. Biol. Chem. 279, 42970-42976). These results strongly suggest that the membrane-proximal region of Htr proteins interact with their cognate sensory rhodopsin cytoplasmic domains as part of the signal-relay coupling between the proteins.

Archaeal Proteins↗

PubMed2004

Characterization of RS29, a blue-green proteorhodopsin variant from the Red Sea.

Using structural modeling comparisons and mutagenesis, amino acid residue 105 was found to function as a spectral tuning switch in marine proteorhodopsins (PR). Changes at this position account for most of the spectral difference between blue-absorbing PRs (B-PRs), and green-absorbing PRs (G-PRs). Here we analyzed a Red Sea variant (RS29) from a new family of PRs that is composed of G-PR type variants that possess glutamine instead of leucine at position 105 like in B-PRs. The absorption spectrum as well as photocycle of RS29 variant were measured and compared to point-mutated 'position 105' PRs. Unexpectedly, the absorption maximum of RS29 was 515 nm, a smaller blue shift compared to the 498 nm maximum of G-PR_L105Q. We found that two additional residues at positions 65 and 70 each contribute a small red shift to the absorption spectrum of G-PR and therefore appear to account for the intermediate absorption maximum of RS29 by their opposing influences on the spectrum. Our results show that in addition to the retinal pocket position 105 determinant, other residues predicted to be outside the retinal pocket fine-tune the absorption spectra of marine PRs. The RS29 photochemical reaction cycle was found to be 2 orders of magnitude slower than that of G-PR with a t(1/2) of >600 ms. This result raises the possibility of regulatory (i.e. sensory) rather than energy harvesting functions for some members of the PR family.

Base Sequence↗

PubMed2004

Light-induced intramolecular charge movements in microbial rhodopsins in intact E. coli cells.

Microbial rhodopsins undergo cyclic photochemical reactions (photocycles) in which proton transfers and conformational changes result in charge displacements during transitions between photocycle intermediates. We report a new photoelectric method to monitor charge movements during rhodopsin photocycling with fast kinetic resolution in suspensions of intact E. coli cells. The method monitors electrical currents resulting from asymmetric photoexcitation of microbial rhodopsins by a unilateral laser flash, and kinetically resolves intramolecular charge movements. We investigated E. coli-expressed proton-transporting rhodopsins, specifically green- and blue-absorbing proteorhodopsins (GPR and BPR, respectively) from uncultivated marine plankton, and sensory rhodopsins, namely receptors from Natronomonas pharaonis and Anabaena (Nostoc) sp. PCC7120. Kinetic components of the currents correlate with photochemical transformations of the pigments, and the integrated current measures net transport by the proton-pumping rhodopsins. The photoelectric measurements distinguish between known light-driven transporters and photosensors, and reveal differences in proton transfer reactions in the two tested proton pumps. Screening of nine newly identified proteorhodopsins reveals two with GPR-type charge movements, five with BPR-type, and two with the characteristics of the sensory rhodopsins. The approach developed in the present work provides a direct, rapid and informative method for studying electrogenic events in rhodopsin photocycles and also gives a clue to functions of newly found microbial rhodopsins in nature.

Anabaena↗

PubMed2004

Chlamydomonas sensory rhodopsins A and B: cellular content and role in photophobic responses.

Two retinylidene proteins, CSRA and CSRB, have recently been shown by photoelectrophysiological analysis of RNAi-transformants to mediate phototaxis signaling in Chlamydomonas reinhardtii. Here we report immunoblot detection of CSRA and CSRB apoproteins in C. reinhardtii cells enabling assessment of the cellular content of the receptors. We obtain 9 x 10(4) CSRA and 1.5 x 10(4) CSRB apoprotein molecules per cell in vegetative cells of the wild-type strain 495, a higher value than that for functional receptor cellular content estimated previously from photosensitivity measurements and retinal extraction yields. Exploiting our ability to control the CSRA/CSRB ratio by transformation with receptor gene-directed RNAi, we report analysis of the CSRA and CSRB roles in the photophobic response of the organism by action spectroscopy with automated cell tracking/motion analysis. The results show that CSRA and CSRB each mediate the photophobic swimming response, a second known retinal-dependent photomotility behavior in C. reinhardtii. Due to the different light saturation and spectral properties of the two receptors, CSRA is dominantly responsible for photophobic responses, which appear at high light intensity.

Animals↗