Disruption in murine Eml1 perturbs retinal lamination during early development.

During mammalian development, establishing functional neural networks in stratified tissues of the mammalian central nervous system depends upon the proper migration and positioning of neurons, a process known as lamination. In particular, the pseudostratified neuroepithelia of the retina and cerebrocortical ventricular zones provide a platform for progenitor cell proliferation and migration. Lamination defects in these tissues lead to mispositioned neurons, disrupted neuronal connections, and abnormal function. The molecular mechanisms necessary for proper lamination in these tissues are incompletely understood. Here, we identified a nonsense mutation in the Eml1 gene in a novel murine model, tvrm360, displaying subcortical heterotopia, hydrocephalus and disorganization of retinal architecture. In the retina, Eml1 disruption caused abnormal positioning of photoreceptor cell nuclei early in development. Upon maturation, these ectopic photoreceptors possessed cilia and formed synapses but failed to produce robust outer segments, implying a late defect in photoreceptor differentiation secondary to mislocalization. In addition, abnormal positioning of Müller cell bodies and bipolar cells was evident throughout the inner neuroblastic layer. Basal displacement of mitotic nuclei in the retinal neuroepithelium was observed in tvrm360 mice at postnatal day 0. The abnormal positioning of retinal progenitor cells at birth and ectopic presence of photoreceptors and secondary neurons upon maturation suggest that EML1 functions early in eye development and is crucial for proper retinal lamination during cellular proliferation and development.

[The implantable automatic defibrillator]. / Défibrillation automatique implantable. A propos de quelques avancées techniques récentes.

Technical advances in the design of implantable automatic defibrillators have been constant since the introduction of these devices in the mid 80s. The most obvious advance is the miniaturisation of the devices from which all components have benefited. The capacity of the batteries has improved inversely proportionally to their size, even if the longevity has not always lived up to expectations. The volumic energy of the condensers has improved and their technology also, and their size has decreased. Condensers are still usually made by the electrolytic/aluminium method but tantalum technology is bound to become more generalised because it presents so many advantages. Above all, the circuitry has benefited from the progress of micro-electronics, associating miniaturisation with an increase in more and more complex functions...but requiring more electrical current. Of these functions, algorithms to detect arrhythmias has reduced the number of inappropriate shocks but do not yet have excellent specificity either in single or in dual chamber sensing. Defibrillators incorporating a multisite anti-bradycardiac function are more and more popular because of the close relationship between cardiac failure and sudden death.

The cobY gene of the archaeon Halobacterium sp. strain NRC-1 is required for de novo cobamide synthesis.

Genetic and nutritional analyses of mutants of the extremely halophilic archaeon Halobacterium sp. strain NRC-1 showed that open reading frame (ORF) Vng1581C encodes a protein with nucleoside triphosphate:adenosylcobinamide-phosphate nucleotidyltransferase enzyme activity. This activity was previously associated with the cobY gene of the methanogenic archaeon Methanobacterium thermoautotrophicum strain DeltaH, but no evidence was obtained to demonstrate the direct involvement of this protein in cobamide biosynthesis in archaea. Computer analysis of the Halobacterium sp. strain NRC-1 ORF Vng1581C gene and the cobY gene of M. thermoautotrophicum strain DeltaH showed the primary amino acid sequence of the proteins encoded by these two genes to be 35% identical and 48% similar. A strain of Halobacterium sp. strain NRC-1 carrying a null allele of the cobY gene was auxotrophic for cobinamide-GDP, a known intermediate of the late steps of cobamide biosynthesis. The auxotrophic requirement for cobinamide-GDP was corrected when a wild-type allele of cobY was introduced into the mutant strain, demonstrating that the lack of cobY function was solely responsible for the observed block in cobamide biosynthesis in this archaeon. The data also show that Halobacterium sp. strain NRC-1 possesses a high-affinity transport system for corrinoids and that this archaeon can synthesize cobamides de novo under aerobic growth conditions. To the best of our knowledge this is the first genetic and nutritional analysis of cobalamin biosynthetic mutants in archaea.

Thermodynamic stability of the bacteriorhodopsin lattice as measured by lipid dilution.

To determine the strength of noncovalent interactions that stabilize a membrane protein complex, we have developed an in vitro method for quantifying the dissociation of the bacteriorhodopsin (BR) lattice, a naturally occurring two-dimensional crystal. A lattice suspension was titrated with a short- and long-chain phosphatidylcholine mixture to dilute BR within the lipid bilayer. The fraction of BR in the lattice form as a function of added lipid was determined by visible circular dichroism spectroscopy and fit with a cooperative self-assembly model to obtain a critical concentration for lattice assembly. Critical concentration values of wild-type and mutant proteins were used to calculate the change in lattice stability upon mutation (DeltaDeltaG). By using this method, a series of mutant proteins was examined in which residues at the BR-BR interface were replaced with smaller amino acids, either Ala or Gly. Most of the mutant lattices were destabilized, with DeltaDeltaG values of 0.2-1.1 kcal/mol at 30 degrees C, consistent with favorable packing of apolar residues in the membrane. One mutant, I45A, was stabilized by approximately 1.0 kcal/mol, possibly due to increased lipid entropy. The DeltaDeltaG values agreed well with previous in vivo measurements, except in the case of I45A. The ability to measure the change in stability of mutant protein complexes in a lipid bilayer may provide a means of determining the contributions of specific protein-protein and protein-lipid interactions to membrane protein structure.

brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon.

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176-12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology.

Genome sequence of Halobacterium species NRC-1.

We report the complete sequence of an extreme halophile, Halobacterium sp. NRC-1, harboring a dynamic 2,571,010-bp genome containing 91 insertion sequences representing 12 families and organized into a large chromosome and 2 related minichromosomes. The Halobacterium NRC-1 genome codes for 2,630 predicted proteins, 36% of which are unrelated to any previously reported. Analysis of the genome sequence shows the presence of pathways for uptake and utilization of amino acids, active sodium-proton antiporter and potassium uptake systems, sophisticated photosensory and signal transduction pathways, and DNA replication, transcription, and translation systems resembling more complex eukaryotic organisms. Whole proteome comparisons show the definite archaeal nature of this halophile with additional similarities to the Gram-positive Bacillus subtilis and other bacteria. The ease of culturing Halobacterium and the availability of methods for its genetic manipulation in the laboratory, including construction of gene knockouts and replacements, indicate this halophile can serve as an excellent model system among the archaea.

Structural determinants of purple membrane assembly.

The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of Halobacterium salinarum. High-resolution structural studies, in conjunction with detailed knowledge of the lipid composition, make the purple membrane one of the best models for elucidating the forces that are responsible for the assembly and stability of integral membrane protein complexes. In this review, recent mutational efforts to identify the structural features of bacteriorhodopsin that determine its assembly in the purple membrane are discussed in the context of structural, calorimetric and reconstitution studies. Quantitative evidence is presented that interactions between transmembrane helices of neighboring bacteriorhodopsin molecules contribute to purple membrane assembly. However, other specific interactions, particularly between bacteriorhodopsin and lipid molecules, may provide the major driving force for assembly. Elucidating the molecular basis of protein-protein and protein-lipid interactions in the purple membrane may provide insights into the formation of integral membrane protein complexes in other systems.

Ordered membrane insertion of an archaeal opsin in vivo.

The prevailing model of polytopic membrane protein insertion is based largely on the in vitro analysis of polypeptide chains trapped during insertion by arresting translation. To test this model under conditions of active translation in vivo, we have used a kinetic assay to determine the order and timing with which transmembrane segments of bacterioopsin (BO) are inserted into the membrane of the archaeon Halobacterium salinarum. BO is the apoprotein of bacteriorhodopsin, a structurally well characterized protein containing seven transmembrane alpha-helices (A-G) with an N-out, C-in topology. H. salinarum strains were constructed that express mutant BO containing a C-terminal His-tag and a single cysteine in one of the four extracellular domains of the protein. Cysteine translocation during BO translation was monitored by pulse-chase radiolabeling and rapid derivatization with a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The results show that the N-terminal domain, the BC loop, and the FG loop are translocated in order from the N terminus to the C terminus. Translocation of the DE loop could not be examined because cysteine mutants in this region did not yield a gel shift. The translocation order was confirmed by applying the assay to mutant proteins containing two cysteines in separate extracellular domains. Comparison of the translocation results with in vivo measurements of BO elongation indicated that the N-terminal domain and the BC loop are translocated cotranslationally, whereas the FG loop is translocated posttranslationally. Together, these results support a sequential, cotranslational model of archaeal polytopic membrane protein insertion in vivo.

Homologous gene knockout in the archaeon Halobacterium salinarum with ura3 as a counterselectable marker.

To facilitate the functional genomic analysis of an archaeon, we have developed a homologous gene replacement strategy for Halobacterium salinarum based on ura3, which encodes the pyrimidine biosynthetic enzyme orotidine-5'-monophosphate decarboxylase. H. salinarum was shown to be sensitive to 5-fluoroorotic acid (5-FOA), which can select for mutations in ura3. A spontaneous 5-FOA-resistant mutant was found to contain an insertion in ura3 and was a uracil auxotroph. Integration of ura3 at the bacterioopsin locus (bop ) of this mutant restored 5-FOA sensitivity and uracil prototrophy. Parallel results were obtained with a Deltaura3 strain constructed by gene replacement and with derivatives of this strain in which ura3 replaced bop. These results show that H. salinarum ura3 encodes functional orotidine-5'-monophosphate decarboxylase. To demonstrate ura3-based gene replacement, a Deltabop strain was constructed by transforming a Deltaura3 host with a bop deletion plasmid containing a mevinolin resistance marker. In one approach, the host contained intact ura3 at the chromosomal bop locus; in another, ura3 was included in the plasmid. Plasmid integrants selected with mevinolin were resolved with 5-FOA, yielding Deltabop recombinants at a frequency of > 10-2 in both approaches. These studies establish an efficient new genetic strategy towards the systematic knockout of genes in an archaeon.

An improved tripod amphiphile for membrane protein solubilization.

Intrinsic membrane proteins represent a large fraction of the proteins produced by living organisms and perform many crucial functions. Structural and functional characterization of membrane proteins generally requires that they be extracted from the native lipid bilayer and solubilized with a small synthetic amphiphile, for example, a detergent. We describe the development of a small molecule with a distinctive amphiphilic architecture, a "tripod amphiphile," that solubilizes both bacteriorhodopsin (BR) and bovine rhodopsin (Rho). The polar portion of this amphiphile contains an amide and an amine-oxide; small variations in this polar segment are found to have profound effects on protein solubilization properties. The optimal tripod amphiphile extracts both BR and Rho from the native membrane environments and maintains each protein in a monomeric native-like form for several weeks after delipidation. Tripod amphiphiles are designed to display greater conformational rigidity than conventional detergents, with the long-range goal of promoting membrane protein crystallization. The results reported here represent an important step toward that ultimate goal.

Membrane insertion kinetics of a protein domain in vivo. The bacterioopsin n terminus inserts co-translationally.

The pathway by which segments of a polytopic membrane protein are inserted into the membrane has not been resolved in vivo. We have developed an in vivo kinetic assay to examine the insertion pathway of the polytopic protein bacterioopsin, the apoprotein of Halobacterium salinarum bacteriorhodopsin. Strains were constructed that express the bacteriorhodopsin mutants I4C:H(6) and T5C:H(6), which carry a unique Cys in the N-terminal extracellular domain and a polyhistidine tag at the C terminus. Translocation of the N-terminal domain was detected using a membrane-impermeant gel shift reagent to derivatize the Cys residue of nascent radiolabeled molecules. Derivatization was assessed by gel electrophoresis of the fully elongated radiolabeled population. The time required to translocate and fully derivatize the Cys residues of I4C:H(6) and T5C:H(6) is 46 +/- 9 and 61 +/- 6 s, respectively. This is significantly shorter than the elongation times of the proteins, which are 114 +/- 26 and 169 +/- 16 s, respectively. These results establish that translocation of the bacterioopsin N terminus and insertion of the first transmembrane segment occur co-translationally and confirm the use of the assay to monitor the kinetics of polytopic membrane protein insertion in vivo.

Role of helix-helix interactions in assembly of the bacteriorhodopsin lattice.

The purple membrane of Halobacterium salinarium is a two-dimensional lattice of lipids and the integral membrane protein bacteriorhodopsin (BR). To determine whether helix-helix interactions within the membrane core stabilize this complex, we substituted amino acid residues at the helix-helix interface between BR monomers and examined the assembly of the protein into the lattice. Lattice assembly was demonstrated to fit a cooperative self-assembly model that exhibits a critical concentration in vivo. Using this model as the basis for a quantitative assay of lattice stability, bulky substitutions at the helix-helix interface between BR monomers within the membrane core were shown to be destabilizing, probably due to steric clash. Ala substitutions of two residues at the helix-helix interface also reduced stability, suggesting that the side chains of these residues participate in favorable van der Waals packing interactions. However, the stabilizing interactions were restricted to a small region of the interface, and most of the substitutions had little effect. Thus, the contribution of helix-helix interactions to lattice stability appears limited, and favorable interactions between other regions of neighboring BR monomers or between BR and lipid molecules must also contribute.

Intramembrane substitutions in helix D of bacteriorhodopsin disrupt the purple membrane.

The Halobacterium salinarium purple membrane is a two-dimensional crystalline lattice containing bacteriorhodopsin (BR) and lipid. To test whether molecular packing within the lipid bilayer influences the structural stability of the lattice, BR mutants substituted on the membrane-embedded surface of the protein were expressed in H. salinarium. Lattice stability was assessed by equilibrium density centrifugation of cell lysates containing similar amounts of BR. BR was distributed in low (1.12 to 1.15 g/ml) and high density (1.18 g/ml) membrane fractions. The high density fraction comprised 89% of the total BR in wild-type, but only 19% (G113L), 29% (I117A), 52% (G116L) and 79% (I117F) in the mutants. In each case, this fraction contained BR in a lattice form: its absorption maximum was blue-shifted by < or = 4 nm relative to the wild-type lattice, its light-dark difference spectrum was normal, and its circular dichroism spectrum retained a bilobed feature characteristic of the lattice. Thus, the substitutions do not significantly alter the tertiary structure of the protein. In the low density fraction, the absorption maximum of BR was blue-shifted by 2 to 4 nm relative to the corresponding high density fraction, and the bilobed circular dichroism feature was attenuated (I117F and G116L) or absent (G113L and I117A). This suggests that the substitutions disrupt lattice stability, causing an accumulation of BR monomers or small aggregates. These results support a model in which the BR lattice is stabilized by hydrophobic packing at specific protein-protein and protein-lipid interfaces within the membrane bilayer.

Proton transport by a bacteriorhodopsin mutant, aspartic acid-85-->asparagine, initiated in the unprotonated Schiff base state.

At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-85 replaced by asparagine, is in a yellow form (lambda max approximately 405 nm) with a deprotonated Schiff base. This state resembles the M intermediate of the wild-type photocycle. We used time-resolved methods to show that this yellow form of D85N, which has an initially unprotonated Schiff base and which lacks the proton acceptor Asp-85, transports protons in the same direction as wild type when excited by 400-nm flashes. Photoexcitation leads in several milliseconds to the formation of blue (630 nm) and purple (580 nm) intermediates with a protonated Schiff base, which decay in tens of seconds to the initial state (400 nm). Experiments with pH indicator dyes show that at pH 7, 8, and 9, proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of proton movement is from the cytoplasmic to the extracellular side with major components on the millisecond and second time scales. The slowest electrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state. Transport thus occurs in two steps. In the first step (milliseconds), the Schiff base is protonated by proton uptake from the cytoplasmic side, thereby forming the blue state. From the pH dependence of the amplitudes of the electrical and photocycle signals, we conclude that this reaction proceeds in a similar way as in wild type--i.e., via the internal proton donor Asp-96. In the second step (seconds) the Schiff base deprotonates, releasing the proton to the extracellular side.

Rapid high-yield purification and liposome reconstitution of polyhistidine-tagged sensory rhodopsin I.

We have used Ni(2+)-affinity chromatography as a rapid and efficient method to purify a sensory rhodopsin I (SR-I) derivative containing six consecutive histidine residues at its C-terminus (His-tagged SR-I). The protein was expressed in Halobacterium salinarium by integrating the corresponding gene at the chromosomal bacterioopsin locus under the control of the bacterioopsin promoter. His-tagged SR-I retains native SR-I photochemical reactions in purified membranes and phototaxis signaling function in vivo. Immobilized Ni(2+)-affinity chromatography of membranes solubilized in 1% layryl maltoside provides a single-step purification of the protein to electrophoretic homogeneity (> or = 90% pure). The procedure yields 1.7 mg pure photoactive protein/liter of culture (60% efficiency). This yield combined with engineered overproduction of the protein provides at least 120-fold greater amounts than that of a previously reported multistep purification procedure, permitting structural and biochemical analysis previously not feasible. The purified protein in lauryl maltoside at pH 5.3 exhibits a visible absorption maximum at 587 nm characteristic of SR-I. Spectrometric titration reveals an alkaline-induced species at 550 nm previously observed with transducer-free SR-I in native membranes. A previously unreported structured absorption band at 400 nm, consistent with a deprotonated Schiff base, forms with the same pKa as the 550-nm species. His-tagged SR-I reconstituted into phosphatidylglycerol proteoliposomes retains properties of transducer-free SR-I in native membranes: its flash-induced absorption difference spectrum is identical, its photochemical reaction cycle kinetics show a similar pH dependence, and it forms a photoactive 550-nm species under alkaline conditions. These results indicate His-tagged SR-I reconstituted in proteoliposomes is suitable for analyzing SR-I interaction with its transducer protein in vitro.

Intramolecular charge transfer in the bacteriorhodopsin mutants Asp85-->Asn and Asp212-->Asn: effects of pH and anions.

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->Asn and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, both in the presence of Cl- (150 mM) and in its absence (75 mM SO4(2-)). Under both conditions the color of the pigment is blue (lambda max = 615 nm). The time course of the photovoltage kinetics is similar to that of the acid-blue form of wild-type, except that an additional transient charge motion occurs with time constants of 60 microseconds and 1.3 ms, indicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is suggested that this is the group XH, which is responsible for proton release in wild-type. At pH 1, the photovoltage signal of D85N changes upon the addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore, the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pKa of the Schiff base, there is a mixture of "M-like" and "N-like" states. Net proton transport in the same direction as in wild-type is restored in D85N from this N-like state. With the mutant Asp212-->Asn (D212N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire pH range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage experiments indicate that net proton pumping is restored. However, this Cl(-)-induced activation of net charge-transport in D212N is only partial. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chloride-dependent features as those accompanying the acid-blue to acid-purple transition of the wild-type.

Characterization of the Tn5 transposase and inhibitor proteins: a model for the inhibition of transposition.

Tn5 is a composite transposon consisting of two IS50 sequences in inverted orientation with respect to a unique, central region encoding several antibiotic resistances. The IS50R element encodes two proteins in the same reading frame which regulate the transposition reaction: the transposase (Tnp), which is required for transposition, and an inhibitor of transposition (Inh). The inhibitor is a naturally occurring deletion variant of Tnp which lacks the N-terminal 55 amino acids. In this report, we present the purification of both the Tnp and Inh proteins and an analysis of their DNA binding properties. Purified Tnp, but not Inh, was found to bind specifically to the outside end of Tn5. Inh, however, stimulated the binding activity of Tnp to outside-end DNA and was shown to be present with Tnp in these bound complexes. Inh was also found to exist as a dimer in solution. These results indicate that the N-terminal 55 amino acids of Tnp are required for sequence-specific binding. They also suggest that Inh inhibits transposition by forming mixed oligomers with Tnp which still bind to the ends of the transposon but are defective for later stages of the transposition reaction.

X-ray diffraction of a cysteine-containing bacteriorhodopsin mutant and its mercury derivative. Localization of an amino acid residue in the loop of an integral membrane protein.

We have used heavy-atom labeling and X-ray diffraction to localize a single amino acid in the integral membrane protein bacteriorhodopsin (bR). To provide a labeling site, we used the bR mutant, A103C, which contains a unique cysteine residue in the short loop between transmembrane alpha-helices C and D. The mutant protein was expressed in and purified from Halobacterium halobium, where it forms a two-dimensional crystalline lattice. In the lattice form, the protein reacted with the sulfhydryl-specific reagent p-chloromercuribenzoate (p-CMB) in a 1:0.9 stoichiometry to yield the p-mercuribenzoate derivative (A103C-MB). The functional properties of A103C and A103C-MB, including the visible absorption spectrum, light-dark adaptation, photocycle, and proton release kinetics, were similar to those of wild-type bR. X-ray diffraction experiments demonstrated that A103C and A103C-MB membranes have the same hexagonal protein lattice as wild-type purple membrane. Thus, neither the cysteine substitution nor mercury labeling detectably affected bR structure or function. By using Fourier difference methods, the in-plane position of the mercuribenzoate label was calculated from intensity differences in the X-ray diffraction patterns of A103C and A103C-MB. This analysis revealed a well-defined mercury peak located between alpha-helices C and D. The approach reported here offers promise for refining the bR structural model, for monitoring conformational changes in bR photointermediates, and for studying the structure of other proteins in two-dimensional crystals.

Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium.

We have designed, synthesized, and expressed in Halobacterium halobium a gene encoding sensory rhodopsin I (SR-I). The gene has been optimized for cassette mutagenesis by incorporating 30 unique restriction sites with uniform spacing throughout the 720-bp coding region. For expression, the coding region was placed downstream of the promoter and translation initiation region of the bacterioopsin gene on a selectable vector. This construct encodes SR-I with an extended N terminus that includes the 13-amino acid leader sequence and the 8-amino acid N terminus of bacterioopsin. To obtain a SR-I- H. halobium strain for expressing the synthetic gene, we used homologous recombination to delete the chromosomal gene encoding SR-I, sopI. The deletion strain was transformed with the synthetic sopI expression vector. Using antibody directed against the C-terminal region of SR-I, we detected in transformant membranes a protein with the electrophoretic mobility expected for SR-I with a processed N-terminal extension. The synthetic gene product was functionally identical to SR-I. Its flash-induced absorption difference spectrum and photochemical reaction cycle in membrane envelope vesicles were characteristic of SR-I. The protein fully restored phototaxis responses in the deletion strain.