Activation of CK1ɛ by PP2A/PR61ɛ is required for the initiation of Wnt signaling.

This corrects the article DOI: 10.1038/onc.2016.209.

Activation of CK1ɛ by PP2A/PR61ɛ is required for the initiation of Wnt signaling.

Canonical Wnt signaling induces the stabilization of ß-catenin, its translocation to the nucleus and the activation of target promoters. This pathway is initiated by the binding of Wnt ligands to the Frizzled receptor, the association of the LRP5/6 co-receptor and the formation of a complex comprising Dvl-2, Axin and protein kinases CK1α, ɛ, γ and GSK3. Among these, activation of CK1ɛ, constitutively bound to LRP5/6 through p120-catenin, is required for the association of the rest of the components. We describe here that CK1ɛ is activated by the PP2A/PR61ɛ phosphatase. Binding of Wnt ligands promotes the interaction of LRP5/6-associated CK1ɛ with Frizzled-bound PR61ɛ regulatory subunit, facilitating the access of PP2A catalytic subunit to CK1ɛ and its activation, what enables the recruitment of Dvl-2 to the receptor complex and the initiation of the Wnt pathway. Our results uncover the mechanism of activation of the canonical Wnt pathway by its ligands.

Akt2 interacts with Snail1 in the E-cadherin promoter.

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action.

Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.

Beta-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets beta-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of beta-catenin in melanoma cells expressing proteasome-resistant beta-catenin, as well as the effects that perturbation of beta-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity. Beta-catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with beta-catenin without perturbing either LAR or beta-catenin levels or LAR/beta-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of beta-catenin and increased beta-catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclear beta-catenin level and inhibited beta-catenin-driven transcription, as assessed using two different beta-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two beta-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the beta-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant beta-catenin is regulated further by geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.

Regulation of beta-catenin structure and activity by tyrosine phosphorylation.

beta-Catenin plays a dual role as a key effector in the regulation of adherens junctions and as a transcriptional coactivator. Phosphorylation of Tyr-654, a residue placed in the last armadillo repeat of beta-catenin, decreases its binding to E-cadherin. We show here that phosphorylation of Tyr-654 also stimulates the association of beta-catenin to the basal transcription factor TATA-binding protein. The structural bases of these different affinities were investigated. Our results indicate that the beta-catenin C-terminal tail interacts with the armadillo repeat domain, hindering the association of the armadillo region to the TATA-binding protein or to E-cadherin. Phosphorylation of beta-catenin Tyr-654 decreases armadillo-C-terminal tail association, uncovering the last armadillo repeats. In a C-terminal-depleted beta-catenin, the presence of a negative charge at Tyr-654 does not affect the interaction of the TATA-binding protein to the armadillo domain. However, in the case of E-cadherin, the establishment of ion pairs dominates its association with beta-catenin, and its binding is greatly dependent on the absence of a negative charge at Tyr-654. Thus, phosphorylation of Tyr-654 blocks the Ecadherin-beta-catenin interaction, even though the steric hindrance of the C-tail is no longer present. These results explain how phosphorylation of beta-catenin in Tyr-654 modifies the tertiary structure of this protein and the interaction with its different partners.

Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis.

The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent.

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation.

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60(c-src) efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60(c-src), only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.

Nucleotide and Mg2+ dependency of the thermal denaturation of mitochondrial F1-ATPase.

The influence of adenine nucleotides and Mg2+ on the thermal denaturation of mitochondrial F1-ATPase (MF1) was analyzed. Differential scanning calorimetry in combination with ATPase activity experiments revealed the thermal unfolding of MF1 as an irreversible and kinetically controlled process. Three significant elements were analyzed during the thermal denaturation process: the endothermic calorimetric transition, the loss of ATP hydrolysis activity, and the release of tightly bound nucleotides. All three processes occur in the same temperature range, over a wide variety of conditions. The purified F1-ATPase, which contains three tightly bound nucleotides, denatures at a transition temperature (Tm) of 55 degrees C. The nucleotide and Mg2+ content of MF1 strongly influence the thermal denaturation process. First, further binding of nucleotides and/or Mg2+ to MF1 increases the thermal denaturation temperature, whereas the thermal stability of the enzyme is decreased upon removal of the endogenous nucleotides. Second, the stabilizing effect induced by nucleotides is smaller after hydrolysis of ATP (i.e., in the presence of ADP . Mg2+) than under nonhydrolytical conditions (i.e., absence of Mg2+ or using the nonhydrolyzable analog 5'-adenylyl-imidodiphosphate). Third, whereas the thermal denaturation of MF1 fully loaded with nucleotides follows an apparent two-state kinetic process, denaturation of MF1 with a low nucleotide content follows more complex kinetics. Nucleotide content is therefore an important factor in determining the thermal stability of the MF1 complex, probably by strengthening existing intersubunit interactions or by establishing new ones.

Experimental and theoretical characterization of the high-affinity cation-binding site of the purple membrane.

Binding of Mn2+ or Mg2+ to the high-affinity site of the purple membrane from Halobacterium salinarium has been studied by superconducting quantum interference device magnetometry or by ab initio quantum mechanical calculations, respectively. The binding of Mn2+ cation, in a low-spin state, to the high-affinity site occurs through a major octahedral local symmetry character with a minor rhombic distortion and a coordination number of six. A molecular model of this binding site in the Schiff base vicinity is proposed. In this model, a Mg2+ cation interacts with one oxygen atom of the side chain of Asp85, with both oxygen atoms of Asp212 and with three water molecules. One of these water molecules is hydrogen bonded to both the nitrogen of the protonated Schiff base and the Asp85 oxygen. It could serve as a shuttle for the Schiff base proton to move to Asp85 in the L-M transition.

Effect of nucleotides on the thermal stability and on the deuteration kinetics of the thermophilic F0F1 ATP synthase.

Differential scanning calorimetry has been used to characterize the influence of specific nucleotide binding on the thermal unfolding of the F0F1-type ATP synthase from the thermophilic Bacillus PS3 (TF0F1). The calorimetric trace shows an irreversible and kinetically controlled endothermic transition for TF0F1 in the absence of nucleotides. The thermal denaturation occurs at a transition temperature (t(m)) of 81.7 degrees C. The remarkable thermostability of this enzyme was decreased upon tight binding of Mg2+ x ATP to noncatalytic sites, whereas binding of Mg2+ x ADP increased the temperature at which thermal denaturation occurred. At high temperatures, an exothermic transition due to aggregation processes was also affected by nucleotide binding. With the aim to correlate these thermal effects with possible structural differences among the various forms of TF0F1, Fourier transform infrared spectroscopy was carried out. Hydrogen/deuterium exchange was clearly affected by specific nucleotide occupancy. As illustrated by the total extent of protons exchanged, our results demonstrate that more peptide groups are exposed to the medium in the presence of Mg2+ x ATP than in the presence of Mg2+ x ADP. Therefore, consistent with microcalorimetric data, binding of Mg2+ x ADP induces conformational changes which shield amide protons to more buried hydrogen-bonded structures, whereas binding of Mg2+ x ATP results in a more open or flexible structure.

Liposome solubilization and membrane protein reconstitution using Chaps and Chapso.

The process of liposome solubilization and reconstitution of two transport proteins have been studied using Chaps and Chapso (3-[(3-cholamidopropyl)dimethylammonio]-2- hydroxy-1-propanesulfonate). The solubilization of unilamellar liposomes was followed by absorption experiments and the process was shown to fit well to the three-stage model previously proposed for other detergents. The solubilization parameters have been determined and the detergent to phospholipid ratios at which the lamellar-to-micellar transition initiates and ends were estimated to be 0.21 mol/mol and 0.74 mol/mol, for Chapso and 0.4 mol/mol and 1.04 mol/mol for Chaps, respectively. The best conditions for the incorporation of two membrane proteins, bacteriorhodopsin and the H(+)-ATP synthase from chloroplasts, were analyzed at each step of the solubilization process. After detergent removal, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitutions were obtained by addition of the proteins to completely solubilized lipid-detergent micelles. The use of Chapso and Chaps for membrane protein reconstitution studies provides a reproducible method of achieving active proteoliposomes, homogeneous in size, with a low permeability and thus, well suited for transport measurements.

Functional reconstitution of photosystem I reaction center from cyanobacterium Synechocystis sp PCC6803 into liposomes using a new reconstitution procedure.

Photosystem I reaction center from the cyanobacterium Synechocystis sp PCC6803 was reconstituted into phosphatidylcholine/phosphatidic acid liposomes. Liposomes prepared by reversephase evaporation were treated with various amounts of different detergents and protein incorporation was analyzed at each step of the solubilization process. After detergent removal the activities of the resulting proteoliposomes were measured. The most efficient reconstitution was obtained by insertion of the protein complex into preformed liposomes destabilized by saturating amounts of octylglucoside. In the presence of N-methylphenazonium methosulfate and ascorbic acid, liposomes containing the reaction center catalyzed a light-dependent net H+ uptake as measured by the 9-aminoacridine fluorescence quenching and the pH meter. An important benefit of the new reconstitution procedure is that it produces a homogeneous population of large-size proteoliposomes with a low ionic permeability and with a majority inwardly directed H+ transport activity. In optimal conditions, a light-induced delta pH of about 1.8 units could be sustained at 20 degrees C in the presence of valinomycin. In the absence of valinomycin, a "back-pressure" effect of an electrical transmembrane potential decreased both the rate and the extent of the H+ transport. The reaction center was also co-reconstituted with F0F1 H(+)-ATPases from chloroplasts and from the thermophilic bacterium, PS3. The co-reconstituted system was shown to catalyze a light-dependent phosphorylation which could only be measured in the presence of a high concentration of PSI (low lipid/PSI ratios) while no delta pH could be detected.

Structure and Activity of Membrane Receptors: Modeling and Computational Simulation of Ligand Recognition in a Three-Dimensional Model of the 5-Hydroxytryptamine(1A) Receptor.

A three-dimensional molecular model of the transmembrane domain of the 5-HT(1A) receptor (5-HT(1A)R) is presented in the context of a general strategy for modeling the macromolecular structure of a guanine nucleotide binding, regulatory protein coupled receptor (GPCR). The model of the 5-HT(1A)R rests on the definition of the putative residues of the ligand-binding site guided by criteria based on specific models proposed from structure-activity studies and on published results of modifications of GPCRs using methods of molecular biology. The resulting requirements for matching recognition sites in the agonist-binding pocket define the molecular details of the interaction between the agonist 5-HT and the human 5-HT(1A)R that includes: (1) the interaction between the protonated amine moiety and the conserved negative Asp-116, located in TMH 3; (2) the hydrogen bond between the hydroxyl group and Thr-199, located in TMH 5; and (3) the interaction complex between the aromatic ring portion of the ligand and the neutral form of His-192, located in TMH 5. Results from quantum mechanical calculations of the interaction between an agonist and the proposed recognition pocket of the 5-HT(1A)R model suggest a trigger of the receptor activation mechanism resulting from ligand binding. The antagonist-binding pocket of the human 5-HT(1A)R is inferred from the interaction sites of pindolol with the receptor model: (1) the ionic interaction between the protonated amine of the ligand and the side chain of the conserved Asp-116, located in TMH 3; and (2) the hydrogen bonds between the ether oxygen and the hydroxyl group of the ligand and Asn-385, located in TMH 7. Use of the model is proposed to facilitate the identification of the structural elements of agonists and antagonists that are key for their specific functions, in order to achieve the design of new compounds with predetermined pharmacological properties. Copyright 1996 S. Karger AG, Basel

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin.

Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40 degrees C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitutions were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TFO-F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP x min(-1) x mg TF0F1(-1) in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 2. Relationships between proton motive force and ATP synthesis.

The correlation between the rate of ATP synthesis and light-induced proton flux was investigated in proteoliposomes reconstituted with bacteriorhodopsin and ATP synthase from thermophilic Bacillus PS3. By variation of the actinic light intensity it was found that ATP synthase activity depended in a sigmoidal manner on the amplitude of the transmembrane light-induced pH gradient. Maximal rates of ATP synthesis (up to to 200 nmol ATP x min(-1) x mg protein (-1) were obtained at saturating light intensities under a steady-state pH gradient of about pH 1.25. It was demonstrated that this was the maximal deltapH attainable at 40 degrees C in reconstituted proteoliposomes, due to the feedback inhibition of bacteriorhodopsin by the proton gradient it generates. In the absence of valinomycin, a small but significant transmembrane electrical potential could develop at 40 degrees C, contributing to an increase in the rate of ATP synthesis. The H+/ATP stoichiometry was measured at the static-head (equilibrium) conditions from the ratio of the phosphate potential to the size of the light-induced pH gradient and a value of about four was obtained under the maximal electrochemical proton gradient. Increasing the amount of bacteriorhodopsin in the proteoliposomes at a constant F0F1 concentration led to a large increase in the rate of ATP synthesis whereas the magnitude of delta pH remained the same or, at very high bacteriorhodopsin levels, decreased. Consequently the H+/ATP stoichiometry was found to increase significantly with increasing bacteriorhodopsin content. Reconstitutions with mixtures of native and impaired bacteriorhodopsin (Asp96-->Asn mutated bacteriorhodopsin) further demonstrated that this increase in the coupling efficiency could not be related to protein-protein interactions but rather to bacteriorhodopsin donating H+ to the ATP synthase. Increasing the amount of negatively charged phospholipids in the proteoliposomes also increased the coupling efficiency between bacteriorhodopsin and ATP synthase at a constant transmembrane pH gradient. Similar results were obtained with chloroplast ATP synthase. Furthermore, ATP synthase activities induced by delta pH/delta psi transitions were independent of bacteriorhodopsin or anionic lipid levels. These observations were interpreted as indicating that, in bacteriorhodopsin/ATP synthase, proteoliposomes, a localized pathway for coupling light-driven H+ transport by bacteriorhodopsin to ATP synthesis by F0F1 might exist under specific experimental conditions.

Influence of nucleotides on the secondary structure and on the thermal stability of mitochondrial F1 visualized by infrared spectroscopy.

We have studied the secondary structure of mitochondrial F1 using infrared spectroscopy. Our results show that in the absence of added nucleotides this complex contains similar percentages of alpha-helices, beta-structures and reverse turns (30%, 28% and 31%, respectively). The influence of ADP and ATP on the different types of secondary structure was determined; when all the nucleotide-binding sites were occupied, small but reproducible changes were observed, corresponding to a decrease in beta-structure and an increase in alpha-helix and reverse turns. The effect of nucleotide binding on the thermal stability of F1 was also studied; the thermal denaturation temperature, 55 degrees C, was increased by 11 degrees C and 7 degrees C by ATP and ADP, respectively. These results indicate that nucleotide binding affects the secondary structure of F1, stabilizing the complex.

Uv-visible spectroscopy of bacteriorhodopsin mutants: substitution of Arg-82, Asp-85, Tyr-185, and Asp-212 results in abnormal light-dark adaptation.

The light-dark adaptation reactions of a set of bacteriorhodopsin (bR) mutants that affect function and color of the chromophore were examined by using visible absorption spectroscopy. The absorbance spectra of the mutants Arg-82 in equilibrium Ala (Gln), Asp-85 in equilibrium Ala (Asn, Glu), Tyr-185 in equilibrium Phe, and Asp-212 in equilibrium Ala (Asn, Glu) were measured at different pH values during and after illumination. None of these mutants exhibited a normal dark-light adaptation, which in wild-type bR causes a red shift of the visible absorption maximum from 558 nm (dark-adapted bR) to 568 nm (light-adapted bR). Instead a reversible light reaction occurs in the Asp-85 and Asp-212 mutants from a blue form with lambda max near 600 nm to a pink form with lambda max near 480 nm. This light-induced shift explains the appearance of a reversed light adaptation previously observed for the Asp-212 mutants. In the case of the Tyr-185 and Arg-82 mutants, light causes a purple-to-blue transformation similar to the effect of lowering the pH. However, the blue forms observed in these mutants are not identical to those formed by acid titration or deionization of wild-type bR. It is suggested that in all of these mutants, the chromophore has lost the ability to undergo the normal 13-cis, 15-syn to all-trans, 15-anti light-driven isomerization, which occurs in native bR. Instead these mutants may have as stable forms all-trans,syn and 13-cis,anti chromophores, which are not allowed in native bR, except transiently.

Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants. Evidence for two forms of tyrosine-185----phenylalanine.

The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185----Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185----Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a lambda max near 610 nm although the absorption maximum of light-adapted Tyr-185----Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a lambda max near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 microseconds, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.

Substitution of membrane-embedded aspartic acids in bacteriorhodopsin causes specific changes in different steps of the photochemical cycle.

Millisecond photocycle kinetics were measured at room temperature for 13 site-specific bacteriorhodopsin mutants in which single aspartic acid residues were replaced by asparagine, glutamic acid, or alanine. Replacement of aspartic acid residues expected to be within the membrane-embedded region of the protein (Asp-85, -96, -115, or -212) produced large alterations in the photocycle. Substitution of Asp-85 or Asp-212 by Asn altered or blocked formation of the M410 photointermediate. Substitution of these two residues by Glu decreased the amount of M410 formed. Substitutions of Asp-96 slowed the decay rate of the M410 photointermediate, and substitutions of Asp-115 slowed the decay rate of the O640 photointermediate. Corresponding substitutions of aspartic acid residues expected to be in cytoplasmic loop regions of the protein (Asp-36, -38, -102, or -104) resulted in little or no alteration of the photocycle. Our results indicate that the defects in proton pumping which we have previously observed upon substitution of Asp-85, Asp-96, Asp-115, and Asp-212 [Mogi, T., Stern, L. J., Marti, T., Chao, B. H., & Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4148-4152] are closely coupled to alterations in the photocycle. The photocycle alterations observed in these mutants are discussed in relation to the functional roles of specific aspartic acid residues at different stages of the bacteriorhodopsin photocycle and the proton pumping mechanism.

Fourier-transform infrared studies on cation binding to native and modified purple membranes.

Fourier-transform infrared spectroscopy has been used to examine the structural differences in the protein moiety between the native purple and the deionized blue membranes, both at pH 5.0. The spectra demonstrate that deionization of purple membrane decreases the content of the distorted alpha II-helices in favor of the more common alpha I-helices. Changes in the signals from beta-turns are also observed. The changes corresponding to the carboxyl groups suggest that deionization leads to a decrease in the strength of the hydrogen bonds involving carboxyl groups. Most of these effects are reversed progressively upon binding of one to five Mn2+ per bacteriorhodopsin to the deionized membrane. Binding of Hg2+ to the deionized membranes does not restore the purple color but induces global changes similar to, but less intense than, those brought about by Mn2+ binding. However, the effects attributed to the carboxyl groups are opposite to those found for Mn2+. Schiff base reduction or bleaching induces a decrease of the content of the alpha II-helix in favor of the alpha I-helix and a decrease in the strength of hydrogen bonds to carboxyl groups. Deionization of these modified membranes leads to a further loss in the alpha II content. These results indicate a conformational rearrangement of the protein structure between the native purple membrane and the deionized membrane, which could arise from surface potential changes elicited by bound cations. The changes observed in the carboxyl groups suggest that some of them are located structurally close to the retinal environment and may be involved in cation binding.