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J L Spudich

Publications and source records attributed to J L Spudich.

At least 73 records · Page 4Linked to original sources
PubMed1992

Transformation of a bop-hop-sop-I-sop-II-Halobacterium halobium mutant to bop+: effects of bacteriorhodopsin photoactivation on cellular proton fluxes and swimming behavior.

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1992

Sensitivity increase in the photophobic response of Halobacterium halobium reconstituted with retinal analogs: a novel interpretation for the fluence-response relationship and a kinetic modeling.

Phoborhodopsin (also called sensory rhodopsin II) is a photoreceptor protein which mediates photophobic responses of Halobacterium halobium to blue-green light. Under conditions where the synthesis of the chromophore retinal is inhibited, the photophobic system is reconstituted in vivo by incorporation of all-trans retinal or retinal analogs into the apoprotein of phoborhodopsin. Retinal analogs which retard the cyclic photoreaction kinetics of phoborhodopsin increase significantly the sensitivity of the photophobic response. This supports the previously reported hypothesis that signal amplification occurs during the lifetime of intermediate states of the photocycle. The sensitivity increase caused by the chromophore substitution is observed in cells at several different growth stages, i.e. the naturally occurring chromophore (all-trans retinal) does not produce maximal sensitivity at any stage of the culture growth. These results are difficult to interpret in terms of the proposal by Marwan et al. (J. Mol. Biol. 199, 663-664, 1988) that only a single photon is sufficient to cause the photobehavioral response in cells containing native phoborhodopsin. A new interpretation for the fluence-response curves is described based in part on their Poisson statistical analysis. Further, a kinetic model which relates the receptor photochemical reaction cycle to the behavioral response is developed, which accounts for both the sensitivity increase and the shape of the fluence-response curves.

Archaeal Proteins↗
PubMed1992

Absorption and photochemistry of sensory rhodopsin--I: pH effects.

Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH-probe to test whether there is a light-induced proton release to the bulk phase during the photochemical reaction cycle of sensory rhodopsin-I (SR-I). We conclude that the retinylidene Schiff-base proton is retained by SR-I-containing envelope vesicles during the SR-I photocycle under the conditions described here. Bacteriorhodopsin containing vesicles were used as a control to show that light-induced proton release can be observed under identical data acquisition parameters as those used for SR-I-containing vesicles. In addition, the effects of extravesicular pH on the absorption maximum (lambda max) and the SR-I photocycle were studied. SR-I properties are insensitive to pH in the range approximately 3 to approximately 8 with lambda max remaining at 587 nm. The lambda max shifts to 565 nm below pH 3.0 and to 552 nm at pH 10.8 with an apparent pKa of 8.5. Flash-induced absorbance changes of SR-I are described under neutral, alkaline and acidic conditions. The neutral, alkaline and acid SR-I forms each undergo similar photoreactions producing long-lived (> 500 ms decay half-time) blue-shifted intermediates. The UV/near-UV absorption of the photoproducts from neutral and alkaline SR-I indicate a deprotonated Schiff base, whereas acid SR-I produces a species with lambda max > 460 nm indicative of a protonated Schiff base.

Bacteriorhodopsins↗
PubMed1991

Identification of signaling states of a sensory receptor by modulation of lifetimes of stimulus-induced conformations: the case of sensory rhodopsin II.

Lifetimes of stimulus-induced conformations of the phototaxis receptor sensory rhodopsin II (SR-II) from Halobacterium halobium are modulated with seven receptor analogues. By monitoring the receptor dynamics in vitro and physiological responses of the cell in vivo, we observe receptor signaling efficiency increases with decreasing cycling frequency (turnover number) of the receptor. The results demonstrate that modulating lifetimes of protein conformations at the SR-II photoactivation site with chromophore analogues alters the lifetime of the active conformation at the signaling site. We further explore the relationship between photocycle intermediates and the signaling efficiency by analyzing the time-averaged concentrations of the two long-lived spectral intermediates of the SR-II photocycle: S-II350 and S-II530. The results are consistent with the signaling site being activated during formation of S-II350, but not reset by the transition of S-II350 into S-II530; rather deactivation appears to require subsequent decay of S-II530. The results indicate the structural changes at the photoactivation site in the S-II350----S-II530 transition do not reset the signaling site. The procedure used here, applicable in principle to any photoactivated or ligand-activated receptor, provides an initial approach to identify structural alterations key to the receptor activation process.

Archaeal Proteins↗
PubMed1991

Mechanism of activation of sensory rhodopsin I: evidence for a steric trigger.

Sensory rhodopsin I (SR-I) and bacteriorhodopsin (BR) from Halobacterium halobium show broad structural and spectroscopic similarities and yet perform distinct functions: photosensory reception and proton pumping, respectively. Probing the photoactive sites of SR-I and BR with 24 retinal analogs reveals differences in the protein environments near the retinal 13-methyl group and near the beta-ionone ring. 13-cis-Retinal does not form a retinylidene pigment with the SR-I apoprotein, although this isomer binds to the BR apoprotein even more rapidly than all-trans-retinal, the functional isomer of both pigments. The activation of both SR-I and BR requires all-trans/13-cis isomerization of retinal;however, a steric interaction between the retinal 13-methyl group and the protein is required for SR-I activation but not for that of BR. These results reveal a key difference between SR-I and BR that is likely to be the initial diverging point in their photoactivation pathways. We propose the 13-methyl group-protein interaction functions as a trigger for SR-I activation--i.e., converts photon absorption by the chromophore into protein conformational changes. A similar steric trigger is essential for activation of mammalian rhodopsin, indicating a common mechanism for receptor activation in archaebacterial and vertebrate retinylidene photosensors.

Apoproteins↗
PubMed1991

Conformational changes in sensory rhodopsin I: similarities and differences with bacteriorhodopsin, halorhodopsin, and rhodopsin.

FTIR difference spectra have been obtained for the sR587----S373 phototransition of sensory rhodopsin I (sR-I), a signal-transducing protein of Halobacterium halobium. The vibrational modes of the sR587 chromophore have frequencies close to those of the bacteriorhodopsin bR568 chromophore, confirming that the two chromophores have very similar structures and environments. However, the sR-I Schiff base C = N stretch frequency is downshifted relative to bR, consistent with weaker hydrogen bonding with its counterion(s). The carboxyl (COOH) stretch modes of sR-I and halorhodopsin (hR) are at the same frequencies. On the basis of sequence homologies, these bands can be assigned to Asp-106 in helix D and/or Asp-201 in helix G. In contrast, no band was found that could be assigned to the protonation of Asp-76. In bR, the homologous residue Asp-85 serves as the acceptor group for the Schiff base proton. Bands appear in the amide I and II regions at similar frequencies in sR-I, hR, and bR, indicating that despite their different functions they all undergo closely related structural changes. Bands are also detected in the C-H stretch region, possibly due to alterations in the membrane lipids. Similar spectral features are also observed in the lipids of rhodopsin-containing photoreceptor membrane upon light activation.

Amino Acid Sequence↗
PubMed1991

Retinal analog restoration of photophobic responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like chromophore in a eukaryotic rhodopsin.

The strain CC-2359 of the unicellular eukaryotic alga Chlamydomonas reinhardtii originally described as a low pigmentation mutant is found to be devoid of photophobic stop responses to photostimuli over a wide range of light intensities. Photophobic responses of the mutant are restored by exogenous addition of all-trans retinal. We have combined computer-based cell-tracking and motion analysis with retinal isomer and retinal analog reconstitution of CC-2359 to investigate properties of the photophobic response receptor. Most rapid and most complete reconstitution is obtained with all-trans retinal compared to 13-cis, 11-cis, and 9-cis retinal. An analog locked by a carbon bridge in a 6-s-trans conformation reconstitutes whereas the corresponding 6-s-cis locked analog does not. Retinal analogs prevented from isomerization around the 13-14 double bond by a five-membered ring in the polyene chain (locked in either the 13-trans or 13-cis configuration) do not restore the response, but enter the chromophore binding pocket as evidenced by their inhibition of all-trans retinal regeneration of the response. Results of competition experiments between all-trans and each of the 13-locked analogs fit a model in which each chromophore exhibits reversible binding to the photoreceptor apoprotein. A competitive inhibition scheme closely fits the data and permits calculation of apparent dissociation constants for the in vivo reconstitution process of 2.5 x 10(-11) M, 5.2 x 10(-10) M, and 5.4 x 10(-9) M, for all-trans, 13-trans-locked and 13-cis-locked analogs, respectively. The chromophore requirement for the trans configuration and 6-s-trans conformation, and the lack of signaling function from analogs locked at the 13 position, are characteristic of archaebacterial rhodopsins, rather than the previously studied eukaryotic rhodopsins (i.e., visual pigments).

Animals↗
PubMed1991

Evidence that the repellent receptor form of sensory rhodopsin I is an attractant signaling state.

The lifetime of the Halobacterium halobium sensory rhodopsin I (SR-I) photocycle intermediate S373 was modulated by incorporating retinal analogs into SR-I apoprotein in vitro and in vivo. Photocycles by SR-I analog pigments exhibit the same reaction scheme and similar formation rates, but different decay rates, of their S373-like species as monitored by flash spectroscopy in membrane vesicle suspensions. The attractant receptor signaling efficiencies determined by physiological measurements are proportional to the lifetimes of the S373-like intermediates, indicating that S373 is a physiological active conformation (signaling state) of the receptor. A model incorporating this finding into the SR-I photocycle is presented.

Halobacterium↗
PubMed1990

Color regulation in the archaebacterial phototaxis receptor phoborhodopsin (sensory rhodopsin II).

Phoborhodopsin, a repellent phototaxis receptor in Halobacterium halobium, exhibits vibrational fine structure, a feature that has not been identified for any other rhodopsin pigment at physiological temperatures. This conclusion follows form analysis of the absorption properties of the pigment in H. halobium membranes containing native retinal and an array of retinal analogues. The absorption spectrum of the native pigment has a maximum at 487 nm with a pronounced shoulder at 460 nm; however, the bandwidth is that expected for a single retinylidene species. Gaussian band-shape simulation with a spacing corresponding to the vibrational frequencies of polyene stretching modes reproduces the structured absorption spectra of native pigment as well as of analogue phoborhodopsin. Absorption shifts produced by a series of dihydroretinal and other retinal analogues strongly indicate that the dominant factor regulating the color of the pigment is planarization of the retinal ring with respect to the polyene chain.

Archaeal Proteins↗
PubMed1990

Effects of modifications of the retinal beta-ionone ring on archaebacterial sensory rhodopsin I.

Ring desmethyl and acyclic analogues of all-trans retinal were incorporated into the apoprotein of the phototaxis receptor sensory rhodopsin I (SR-I) in Halobacterium halobium membranes. All modified retinals generate SR-I analogue pigments which exhibit "opsin shifts," i.e., their absorption spectra are shifted to longer wavelengths compared with model protonated Schiff bases of the same analogues. Each SR-I pigment analogue exhibits cyclic photochemical reactions as monitored by flash spectroscopy, but the analogue photocycles differ from that of native SR-I by exhibiting pronounced biphasic recovery of flash-induced absorption changes and abnormal flash-induced absorption difference spectra. Despite perturbations in the photochemical properties, the SR-I pigment analogues are capable of both attractant (single photon) and repellent (two photon) phototaxis signaling in cells. Our interpretation is that the hydrophobic ring substituents interact with the binding pocket to maintain the correct configuration for native SR-I absorption and photochemistry, but these interactions are not essential for the physiological function of SR-I as a dual attractant/repellent phototaxis receptor. These results support the conclusion emerging from several studies that the photoactivation process that triggers the conformation changes of SR-I and the related proton pump bacteriorhodopsin is conserved despite the different biological functions of their photoactivation.

Bacteriorhodopsins↗
PubMed1990

All-trans/13-cis isomerization of retinal is required for phototaxis signaling by sensory rhodopsins in Halobacterium halobium.

An analogue of all-trans retinal in which all-trans/13-cis isomerization is blocked by a carbon bridge from C12 to C14 was incorporated into the apoproteins of sensory rhodopsin I (SR-I) and sensory rhodopsin II (SR-II, also called phoborhodopsin) in retinal-deficient Halobacterium halobium membranes. The "all-trans-locked" retinal analogue forms SR-I and SR-II analogue pigments with similar absorption spectra as the native pigments. Blocking isomerization prevents the formation of the long-lived intermediate of the SR-I photocycle (S373) and those of the SR-II photocycle (S-II360 and S-II530). A computerized cell tracking and motion analysis system capable of detecting 2% of native pigment activity was used for assessing motility behavior. Introduction of the locked analogue into SR-I or SR-II apoprotein in vivo did not restore phototactic responses through any of the three known photosensory systems (SR-I attractant, SR-I repellent, or SR-II repellent). We conclude that unlike the phototaxis receptor of Chlamydomonas reinhardtii, which has been reported to mediate physiological responses without specific double-bond isomerization of its retinal chromophore (Foster et al., 1989), all-trans/13-cis isomerization is essential for SR-I and SR-II phototaxis signaling.

Archaeal Proteins↗
PubMed1990

Characterization of Halobacterium halobium mutants defective in taxis.

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

Bacterial Proteins↗
PubMed1989

Sensory rhodopsins I and II modulate a methylation/demethylation system in Halobacterium halobium phototaxis.

This work demonstrates that phototaxis stimuli in the archaebacterium Halobacterium halobium control a methylation/demethylation system in vivo through photoactivation of sensory rhodopsin I (SR-I) in either its attractant or repellent signaling form as well as through the repellent receptor sensory rhodopsin II (SR-II, also called phoborhodopsin). The effects of positive stimuli that suppress swimming reversals (i.e., an increase in attractant or decrease in repellent light) and negative stimuli that induce swimming reversals (i.e., a decrease in attractant or increase in repellent light) through each photoreceptor were monitored by assaying release of volatile [3H]methyl groups. This assay has been used to measure [3H]methanol produced during the process of adaptation to chemotactic stimuli in eubacteria. In H. halobium positive photostimuli produce a transient increase in the rate of demethylation followed by a decrease below the unstimulated value, whereas negative photostimuli cause an increase followed by a rate similar to that of the unstimulated value. Photoactivation of the SR-I attractant and simultaneous photoactivation of the SR-II repellent receptors cancel in their effects on demethylation, demonstrating the methylation system is regulated by an integrated signal. Analysis of mutants indicates that the source for the volatile methyl groups is intrinsic membrane proteins distinct from the chromoproteins that share the membrane. A methyl-accepting protein (94 kDa) previously correlated in amount with the SR-I chromoprotein (25 kDa) is shown here to be missing in a recently isolated SR-I-SR-II+ mutant (Flx3b), thus confirming the association of this protein with SR-I. Photoactivated SR-II in mutant Flx3b controls demethylation, predicting the existence of a photomodulated methyl-accepting component distinct from the 94-kDa protein of SR-I. We present a model in which the three known phototaxis signaling receptor states (the attractant receptor SR-I587, its repellent form S373, and the repellent receptor SR-II490) are coupled to two distinct transducers the demethylation of which is controlled by one integrated signal.

Archaeal Proteins↗
PubMed1988

Membrane potential modulates photocycling rates of bacterial rhodopsins.

Effects of membrane potential on photochemical reactions of three retinal-containing chromoproteins in Halobacterium halobium, sensory rhodopsin I (sR-I), bacteriorhodopsin, and halorhodopsin, are described. Each of the three exhibits a decreased rate of thermal decay of its principal intermediate when photoactivated in an artificially energized compared to a deenergized membrane. The similar response of the three pigments suggests a voltage-dependent conformational change common to their respective photocycles. Spectral and kinetic properties of the sR-I photochemical reaction cycle were measured in phototactic H. halobium cells, and differences from in vitro photocycle kinetics were attributable to the electrical membrane potential present in vivo. In vivo sR-I photocycling rates were reproduced in envelope vesicle preparations in the presence of a valinomycin-induced potassium diffusion potential.

Halobacterium↗
PubMed1988

Fourier transform infrared study of the halorhodopsin chloride pump.

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.

Bacteriorhodopsins↗
PubMed1988

Circular dichroism of halorhodopsin: comparison with bacteriorhodopsin and sensory rhodopsin I.

Circular dichroic (CD) spectra of three related protein pigments from Halobacterium halobium, halorhodopsin (HR), bacteriorhodopsin (BR), and sensory rhodopsin I (SR-I), are compared. In native membranes the two light-driven ion pumps, HR and BR, exhibit bilobe circular dichroism spectra characteristic of exciton splitting in the region of retinal absorption, while the phototaxis receptor, SR-I, exhibits a single positive band centered at the SR-I absorbance maximum. This indicates specific aggregation of protein monomers of HR, as previously noted [Sugiyama, Y., & Mukohata, Y. (1984) J. Biochem. (Tokyo) 96, 413-420], similar to the well-characterized retinal/retinal exciton interaction in the purple membrane. The absence of this interaction in SR-I indicates SR-I is present in the native membrane as monomers or that interactions between the retinal chromophores are weak due to chromophore orientation or separation. Solubilization of HR and BR with nondenaturing detergents eliminates the exciton coupling, and the resulting CD spectra share similar features in all spectral regions from 250 to 700 nm. Schiff-base deprotonation of both BR and HR yields positive CD bands near 410 nm and shows similar fine structure in both pigments. Removal of detergent restores the HR native spectrum. HR differs from BR in that circular dichroic bands corresponding to both amino acid and retinal environments are much more sensitive to external salt concentration and pH. A theoretical analysis of the exciton spectra of HR and BR that provides a range of interchromophore distances and orientations is performed.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1988

Methyl-accepting protein associated with bacterial sensory rhodopsin I.

In vivo radiolabeling of Halobacterium halobium phototaxis mutants and revertants with L-[methyl-3H] methionine implicated seven methyl-accepting protein bands with apparent molecular masses from 65 to 150 kilodaltons (kDa) in adaptation of the organism to chemo and photo stimuli, and one of these (94 kDa) was specifically implicated in phototaxis. The lability of the radiolabeled bands to mild base treatment indicated that the methyl linkages are carboxylmethylesters, as is the case in the eubacterial chemotaxis receptor-transducers. The 94-kDa protein was present in increased amounts in an overproducer of the apoprotein of sensory rhodopsin I, one of two retinal-containing phototaxis receptors in H. halobium. It was absent in a strain that contained sensory rhodopsin II and that lacked sensory rhodopsin I and was also absent in a mutant that lacked both photoreceptors. Based on the role of methyl-accepting proteins in chemotaxis in other bacteria, we suggest that the 94-kDa protein is the signal transducer for sensory rhodopsin I. By [3H]retinal labeling studies, we previously identified a 25-kDa retinal-binding polypeptide that was derived from photochemically reactive sensory rhodopsin I. When H. halobium membranes containing sensory rhodopsin I were treated by a procedure that stably reduced [3H]retinal onto the 25-kDa apoprotein, a 94-kDa protein was also found to be radiolabeled. Protease digestion confirmed that the 94-kDa retinal-labeled protein was the same as the methyl-accepting protein that was suggested above to be the signal transducer for sensory rhodopsin I. Possible models are that the 25- and 94-kDa proteins are tightly interacting components of the photosensory signaling machinery or that both are forms of sensory rhodopsin I.

Bacterial Proteins↗