PubMed Health⌕ Search

Biomedical subjects

J L Spudich

Publications and source records attributed to J L Spudich.

At least 91 records · Page 5Linked to original sources
PubMed1987

The photochemical reactions of bacterial sensory rhodopsin-I. Flash photolysis study in the one microsecond to eight second time window.

Halobacterium halobium Flx mutants are deficient in bacteriorhodopsin (bR) and halorhodopsin (hR). Such strains are phototactic and the light signal detectors are two additional retinal pigments, sensory rhodopsins I and II (sR-I and sR-II), which absorb maximally at 587 and 480 nm, respectively. A retinal-deficient Flx mutant, Flx5R, overproduces sR-I-opsin and does not show any photochemical activity other than that of sR-I after the pigment is regenerated by addition of all-trans retinal. Using native membrane vesicles from this strain, we have resolved a new photointermediate in the sR-I photocycle between the early bathointermediate S610 and the later intermediate S373. The new form, S560, resembles the L intermediate of bR in its position in the photoreaction cycle, its relatively low extinction, and its moderate blue shift. It forms with a half-time of approximately 90 microseconds at 21 degrees C, concomitant with the decay of S610. Its decay with a half-time of 270 microseconds parallels the appearance of S373. From a data set consisting of laser flash-induced absorbance changes (300 ns, 580-nm excitation) measured at 24 wavelengths from 340 to 720 nm in a time window spanning 1 microsecond to 8 s we have calculated the spectra of the photocycle intermediates assuming a unidirectional, unbranched reaction scheme.

Bacteriorhodopsins↗
PubMed1987

Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I.

Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters. The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing). After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca. 2-s period of low reversal frequency before the state N Poisson rate is restored. The stochastic nature of the H. halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously. Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition. Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal. Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities. This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling. In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II).

Bacteriorhodopsins↗
PubMed1986

Properties of a second sensory receptor protein in Halobacterium halobium phototaxis.

A second slow-cycling retinylidene protein, in addition to slow-cycling (sensory) rhodopsin (SR), can be bleached with hydroxylamine and regenerated with all-trans retinal in photosensory signaling Halobacterium halobium membranes. Flash photolysis shows this protein undergoes a photochemical reaction cycle characterized by photoconversion of its ground state (lambda max 480 nm) to a species with lambda max less than or equal to 360 nm, which thermally regenerates the 480-nm species with a t1/2 of 260 msec at 25 degrees C, under conditions in which SR photocycles at 650 msec in the same membranes. Mutants characterized with respect to their phototaxis behavior are identified which contain SR and the 480-nm pigment, the latter ranging from undetectable to a concentration equal to that of SR. Receptor mutants lacking all phototaxis sensitivity lack both of the photochemically reactive proteins. The mutant properties contribute to an accumulation of behavioral and spectroscopic evidence that the 480-nm pigment is a second sensory photoreceptor in H. halobium. NaDodSO4-polyacrylamide gel electrophoresis of [3H]retinal-labeled membrane proteins from the mutants indicates SR and the 480-nm pigment contain distinct chromophoric polypeptides differing in their migration rates. The data implicate polypeptides of 25,000 Mr and 23,000 Mr as retinal-binding polypeptides of SR and the 480-nm protein, respectively.

Bacterial Proteins↗
PubMed1986

Excitation signal processing times in Halobacterium halobium phototaxis.

Phototaxis responses of Halobacterium halobium were monitored with a computerized cell-tracking system coupled to an electronic shutter controlling delivery of photostimuli. Automated analysis of rates of change in direction and linear speeds provided detection of swimming reversals with 67 ms resolution, permitting measurement of distinct phases of the responses to attractant and repellent stimuli. After stimulation, there was a latency period in which the population reversal frequency was unchanged, followed by an excitation phase in which reversal frequency increased, and a slower adaptation phase in which reversal frequency returned to its prestimulus value. A step-decrease in illumination of the attractant receptor slow-cycling or sensory rhodopsin (SR) (lambda max, 587 nm) was interpreted by the cells as an unfavorable stimulus and, after a minimum latency of 0.70 +/- 0.14 s, induced swimming reversals with the peak response occurring 1.34 +/- 0.07 s after onset of the stimulus. Two distinct repellent responses in the near UV/blue were observed. One was a reversal response to 400 nm light, which was dependent on orange-red background illumination as expected for the photointermediate repellent form of SR (lambda max, 373 nm). The minimum latency of this response was approximately the same as that of the SR attractant system. The second was a reversal response with shorter minimum latency (0.40 +/- 0.07 s) to light of longer wavelength (450 nm) than absorbed by the known SR repellent form. This result confirms recent findings of an additional repellent photosystem in this spectral range. Further, the longer wavelength repellent response is independent of orange-red background illumination, indicating that the photoreceptor mediating this response is not a photointermediate of SR.

Bacteriorhodopsins↗
PubMed1986

Chromophore/protein interaction in bacterial sensory rhodopsin and bacteriorhodopsin.

Retinal analogues with altered conjugated double bond systems or altered stereochemistry were incorporated into the phototaxis receptor sensory rhodopsin (SR) and the light-driven proton pump bacteriorhodopsin (BR) from Halobacterium halobium. Wavelength shifts in absorption ("opsin shifts") due to analogue interaction with the protein microenvironment demonstrate that the same overall electrostatic and steric properties of the retinal binding-site structures exist in both proteins despite their different functions. pi-Electron calculations from the opsin shifts lead to a new description of protein charge distribution that applies to the binding sites of both SR and BR. The new data extends the previously proposed external point charge model for BR to include an ion-pair protein/chromophore interaction near the beta-ionone moiety. The new data modifies the previously proposed external point-charge model, the derivation of which involved an experimentally erroneous opsin shift for one of the BR analogues.

Bacteriorhodopsins↗
PubMed1985

Biochemical characterization of halorhodopsin in native membranes.

Procedures are described for selectively radiolabeling the protein moiety (haloopsin) or the chromophoric prosthestic group (retinal) of the light-driven chloride pump halorhodopsin in intact cells of Halobacterium halobium. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autofluorography, two retinal-binding polypeptides are observed to band near the known molecular weight of the halorhodopsin chromophoric polypeptide (25,000). Synthesis of one of these polypeptides is controlled by retinal and is sufficient for generation of complete halorhodopsin function. The other is constitutively produced by the cells and differs chemically from the haloopsin protein as indicated by differences in their V8 protease digestion patterns. V8 protease cleavage of haloopsin in its native membrane is compared with that of the protein in denaturing and nondenaturing detergents. Protease cleavage sites available in the denatured haloopsin molecule are hidden in its native membrane-integrated conformation and in nondenaturing detergent micelles. Treatment with a variety of proteases indicates susceptibility of a short terminal region of the haloopsin chain in its native conformation.

Bacteriorhodopsins↗
PubMed1985

Selection and properties of phototaxis-deficient mutants of Halobacterium halobium.

A method for isolating phototaxis-deficient (Pho-) mutants of Halobacterium halobium was developed. The procedure makes use of a flashing repellent light to induce frequent reversals of swimming direction by responsive cells, thereby impeding their migration along a small capillary and resulting in a spatial separation of the parent population and a population enriched for Pho- cells. Two classes of Pho- mutants were obtained by this selection scheme: those which have lost the chemotactic response (Che-) as well as phototaxis sensitivity (general taxis mutants), and those which are defective in steps specific to phototaxis (photosignaling mutants). In the latter class, several retinal synthesis mutants were isolated, as well as a strain which fit the expected properties of a mutant lacking a functional photoreceptor protein. On the basis of spectroscopic and swimming behavior studies, the retinal-containing protein, slow-cycling or sensory rhodopsin (SR), was previously proposed to be a dual-function sensory receptor mediating both attractant and repellent photosensing. The receptor mutant Pho81 fulfills two predictions which provide direct genetic evidence for this proposal. The mutant has lost SR photoactivity as determined by spectroscopic measurements, and it has simultaneously lost both attractant and repellent phototaxis sensitivity. Comparison of [3H]retinal-labeled membrane proteins from the mutant and its SR-containing parent implicated a 25,000 Mr polypeptide as the chromophoric polypeptide of SR.

Chemotaxis↗
PubMed1984

Bacterial rhodopsins monitored with fluorescent dyes in vesicles and in vivo.

Three retinal-containing pigments have been detected in Halobacterium halobium membranes: bacteriorhodopsin (bR), halorhodopsin (hR), and slow-cycling rhodopsin (sR). The first two hyperpolarize the cell membrane by electrogenic transport of H+ and Cl-, respectively. The third pigment, sR, may be a photosensory receptor since mutants lacking bR and hR retain their retinal-dependent phototaxis responses. We monitored light-induced changes in fluorescence of several voltage-sensitive dyes in cells and membrane vesicles. Red light-induced potential changes generated by bR and hR were similar to signals described previously. Signals generated by hR could be identified using four criteria: wavelength dependence, Cl- dependence, shunting by valinomycin and K+, and the absence of these signals in hR-deficient mutants. The absence (detection limit approximately 0.5 mV) of hyperpolarization signals in bR-hR-sR+ vesicles and cells shows that sR photochemical reactions are nonelectrogenic. Two signals independent of bR and hR were measured: blue light caused a decrease and red light an increase in dye fluorescence. Both signals appear to derive from sR on the basis of their retinal-dependence and action spectra. In a retinal-deficient mutant strain (Flx3R), both sR signals appeared after addition of all-trans retinal. In this strain retinal also restores phototaxis sensitivity within the same time scale. The retinal concentration dependence for all four parameters monitored--the attractant (red) and repellent (blue) phototaxis, and the red light and blue light-induced fluorescence signals--is the same. This correlation is consistent with the hypothesis that both attractant and repellent responses are mediated by sR, as suggested by Bogomolni and Spudich (Proc. Natl. Acad. Sci. USA. 79:6250-6254 (1982)).

Bacteriorhodopsins↗
PubMed1983

Phase-lifetime spectrophotometry of membranes from ion flux mutants of Halobacterium halobium.

Phase-lifetime spectrophotometry has been used to study the rate processes associated with intermediates in the photocycling pigments in membrane vesicles of mutant strains of Halobacterium halobium. Vesicles deficient in bacteriorhodopsin, but containing halorhodopsin, were monitored with light at 490 nm. Two relaxation processes, with kinetic parameters largely independent of pH over the range 6.2-7.8, were found to be associated with halorhodopsin photocycling in 4 M NaCl, 10 mM buffer at 23 degrees C. The average relaxation times are 0.94 and 11.4 ms. When vesicles deficient in both bacteriorhodopsin and halorhodopsin were monitored at 370 nm, a single relaxation process with an average relaxation time of 168 ms was detected. This process is independent of pH over the range 4.7-8.8. Examination of vesicles from ion flux mutants showed this slow process to be unrelated to halorhodopsin content and to derive from another photoreactive retinal pigment, possibly the recently described slow cycling pigment s-rhodopsin.

Bacteriorhodopsins↗
PubMed1983

Genetic and biochemical resolution of the chromophoric polypeptide of halorhodopsin.

Retinal-binding proteins in bacteriorhodopsin-deficient membranes of Halobacterium halobium have been separated on polyacrylamide gels after labeling with (3H)retinal. Four labeled polypeptides are observed: one at an Mr of 19,000, two distinct bands near 25,000, and one at 94,000. Comparing halorhodopsin-containing and halorhodopsin-deficient mutants and using ion translocation measurements and flash spectroscopy to test for the photoactive retinal pigments, we show that the lower of the two radiolabeled bands at 25,000 is the retinal-binding polypeptide of halorhodopsin. The identities of the other three polypeptides are not known, but one of them, probably the upper of the two 25,000 bands, is likely to be s-rhodopsin.

Bacteriorhodopsins↗
PubMed1983

Spectroscopic discrimination of the three rhodopsinlike pigments in Halobacterium halobium membranes.

Membranes of Halobacterium halobium contain two photochemically reactive retinal pigments in addition to the proton pump bacteriorhodopsin. One, halorhodopsin, is also an electrogenic ion pump with a fast (on a scale of milliseconds) photoreaction cycle. The other, s-rhodopsin, is active in the same spectral region, but has a much slower photoreaction cycle (on a scale of seconds). S-rhodopsin is not an electrogenic ion pump and its properties suggest it functions as the receptor pigment for phototaxis. All three pigments have very similar absorption spectra. The recent isolation of mutants deficient in both bacteriorhodopsin and halorhodopsin and in retinal synthesis has allowed us to resolve the absorption spectra of s-rhodopsin and halorhodopsin. At neutral pH s-rhodopsin has an absorption maximum at 587 +/- 2 nm and halorhodopsin at 578 +/- 2 nm. At pH 10.8, lambda max for s-rhodopsin is shifted to 552 nm and extinction decreases slightly (15%) while halorhodopsin loses all extinction above 500 nm. Both effects are fully reversible and allow determination of the amounts of s-rhodopsin and halorhodopsin in membrane preparations containing both pigments. Both pigments were present in earlier studies of H. halobium membranes, and in view of these findings, several observations must be reinterpreted.

Bacteriorhodopsins↗
PubMed1982

Control of transmembrane ion fluxes to select halorhodopsin-deficient and other energy-transduction mutants of Halobacterium halobium.

We describe a selection method for mutants altered in the generation and regulation of transmembrane ion flux in Halobacterium halobium. The method is based on experimental control of ion fluxes by a combination of light, ionophore, and external pH to generate an imbalance in the cells' proton circulation through their membranes. The steady-state proton circulation is increased by the introduction of a small inward proton leak with a protonophore. The cells are then illuminated to excite halorhodopsin, which hyperpolarizes the membrane and drives protons into the cells. As a result, wild-type cells suffer cytoplasmic acidification, which causes a dramatic loss of motility and suppresses their growth. These properties can be used to select for mutants that escape cytoplasmic acidification because either they lack halorhodopsin function or they have a greater capacity to eject protons during the illumination. In a popular selected by this method, 97% of the individual cells were demonstrably altered in ion flux properties. Cells were selected with alterations in the halobacterial rhodopsin, specifically with deficiencies in membrane potential generation by halorhodopsin and with increased cellular proton ejection by bacteriorhodopsin. We describe properties of one of the halorhodopsin-deficient strains, Flx37.

Bacteriological Techniques↗
PubMed1982

Identification of a third rhodopsin-like pigment in phototactic Halobacterium halobium.

Mutant Halobacterium halobium strains deficient in all previously reported rhodopsin-like pigments show phototaxis responses comparable to those of wild-type strains. Spectroscopic analysis reveals the presence of a third retinal-containing pigment in the cells and their membrane fractions. It undergoes a photoreaction cycle with a half-time of approximately equal to 1 sec at room temperature and at physiological light intensities the photostationary state of the pigment consists of two species, one absorbing in the 580- to 590-nm region and the other at 373 nm, both of which are photoactive. Illumination of the long-wavelength species generates the 373-nm intermediate, which upon photoexcitation reconverts to the long-wavelength form. Therefore, changes in the relative light intensities in the long- and short-wavelength regions of the visible spectrum cause opposing shifts in the photostationary state. The spectral sensitivity of this pigment correlates with the color-discriminating phototaxis sensitivities of this organism and strongly suggests that it is the sensory photoreceptor.

Bacteriorhodopsins↗
PubMed1981

Photosensitive phosphoproteins in Halobacteria: regulatory coupling of transmembrane proton flux and protein dephosphorylation.

A photoregulated reversible protein phosphorylation system controlled by the halobacterial rhodopsins was recently reported. The results presented in this paper identify the initial steps in the pathway from the absorption of light to the photoregulated protein phosphorylation and dephosphorylation reactions. Action spectrum, biochemical, and genetic analyses show that the proton pump bacteriorhodopsin mediates light-induced dephosphorylation of three photoregulated phosphoproteins. Light absorbed by bacteriorhodopsin is used to establish a proton efflux from the cells. The increase in the inwardly directed protonmotive force (pmf) from this efflux induces dephosphorylation of the three phosphoproteins, as demonstrated by the effects of the protonophore CCCP and of artificially imposed transmembrane pH gradients. Upon darkening the cells, cessation of the proton efflux through bacteriorhodopsin causes a decrease in pmf, which induces rephosphorylation of the proteins. Pmf appears to function as a regulator rather than a driving force in this system. Measurements of pmf-driven ATP synthesis in our conditions indicate the regulation of protein phosphorylation by pmf is probably not a consequence of proton flux through the H+ ATPase, a known energy coupling structure in these cells. The properties of this system may indicate the existence of a pmf detector which regulates kinase or phosphatase activity; i.e., a regulatory coupling device.

Adenosine Triphosphate↗
PubMed1980

Light-regulated retinal-dependent reversible phosphorylation of Halobacterium proteins.

By (32P)orthophosphate pulse-labeling in vivo, we have identified a class of phosphoproteins in Halobacterium halobium. Exposing labeled whole cells to light results in a rapid dephosphorylation of two of the proteins, which are rapidly rephosphorylated upon darkening of the cells. The light sensitivity of these two proteins is abolished by the retinal synthesis inhibitor nicotine and is restored upon addition of exogenous retinal to the cells. This indicates that the dephosphorylation depends on the rhodopsin-like (retinal-containing) photoreceptors of this organism. Retinal pigments, one of which is the retinyldene protein bacteriorhodopsin, are responsible for both photoenergy and photosensory transduction by Halobacterium. The light-regulated phosphoproteins are of molecular weights of approximately 80,000 and 100,000. Acid and hydroxylamine resistance of the phosphate bonds in both proteins suggests phosphoserine or phosphothreonine linkages.

Bacterial Proteins↗
PubMed1980

Regulation of the Chlamydomonas cell cycle by light and dark.

By growing cells in alternating periods of light and darkness, we have found that the synchronization of phototrophically grown Chlamydomonas populations is regulated at two specific points in the cell cycle: the primary arrest (A) point, located in early G1, and the transition (T) point, located in mid-G1. At the A point, cell cycle progression becomes light dependent. At the T point, completion of the cycle becomes independent of light. Cells transferred from light to dark at cell cycle position between the two regulatory points enter a reversible resting state in which they remain viable and metabolically active, but do not progress through their cycles. The photosystem II inhibitor dichlorophenyldimethylurea (DCMU) mimics the A point block induced by darkness. This finding indicates that the A point block is mediated by a signal that operates through photosynthetic electron transport. Cells short of the T point will arrest in darkness although they contain considerable carbohydrate reserves. After the T point, a sharp increase occurs in starch degradation and in the endogenous respiration rate, indicating that some internal block to the availability of stored energy reserves has now been released, permitting cell cycle progression.

Carbon Dioxide↗
PubMed1979

Specific inactivator of flagellar reversal in Salmonella typhimurium.

Specific inhibition of flagellar rotation reversal was observed after exposure of chemotactic Salmonella typhimurium to citrate autoclaved at neutral pH. The presence of a rotation reversal inactivator was established in autoclaved citrate-containing media and nutrient broth. Since modulation of flagellar rotation by attractants and repellents is the basis of chemotactic behavior, a specific inhibitor of rotation reversal, which is essential for tumble generation, provides a useful probe into the molecular mechanism of bacterial chemotaxis. The inactivator inhibits clockwise rotation without affecting counterclockwise rotation, speed of rotation, or the capacity of the cells to grow and divide. Inactivation of clockwise rotation is gradual and irreversible, differing from the transient inhibition of clockwise rotation by attractants, which is characterized by an immediate suppression followed by a return to normal rotation patterns. The rotation reversal inactivator is stable to acidification, rotary evaporation, lyophilization, and rehydration.

Chemotaxis↗