Transducer protein HtrI controls proton movements in sensory rhodopsin I

Sensory rhodopsin I (SR-I lambda(max) 587 nm) is a phototaxis receptor in the archaeon Halobacterium salinarium. Photoisomerization of retinal in SR-I generates a long-lived intermediate with lambda(max) 373 nm which transmits a signal to the membrane-bound transducer protein HtrI. Although SR-I is structurally similar to the electrogenic proton pump bacteriorhodopsin (BR), early studies showed its photoreactions do not pump protons, nor result in membrane hyperpolarization. These studies used functionally active SR-I, that is, SR-I complexed with its transducer HtrI. Using recombinant DNA methods we have expressed SR-I protein containing mutations in ionizable residues near the protonated Schiff base, and studied wild-type and site-specifically mutated SR-I in the presence and absence of the transducer protein. UV-Vis kinetic absorption spectroscopy, FT-IR, and pH and membrane potential probes reveal transducer-free SR-I photoreactions result in vectorial proton translocation across the membrane in the same direction as that of BR. This proton pumping is suppressed by interaction with transducer which diverts the proton movements into an electroneutral path. A key step in this diversion is that transducer interaction raises the pK(a) of the aspartyl residue in SR-I (Asp76) which corresponds to the primary proton-accepting residue in the BR pump (Asp85). In transducer-free SR-I, our evidence indicates the pK(a) of Asp76 is 7.2, and ionized Asp76 functions as the Schiff base proton acceptor in the SR-I pump. In the SR-I/HtrI complex, the pK(a) of Asp76 is 8.5, and therefore at physiological pH (7.4) Asp76 is neutral. Protonation changes on Asp76 are clearly not required for signaling since the SR-I mutants D76N and D76A are active in phototaxis. The latent proton-translocation potential of SR-I may reflect the evolution of the SR-I sensory signaling mechanism from the proton pumping mechanism of BR.     

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)     

Bioorganic chemistry of the purple membrane ofHalobacterium halobium — Chromophore and apoprotein modified bacteriorhodopsins

Iodophenyl and anthryl retinal analogues have been synthesized. Thetrans-isomers have been isolated and purified by high pressure liquid chromatography. The purified isomers have been further characterized by nuclear magnetic resonance and ultraviolet-visible spectroscopy. Incubation of these retinal analogues with apoprotein (bacterioopsin), isolated from the purple membrane ofHalobacterium halobium gave new bacteriorhodopsin analogues. These analogues have been investigated for their absorption properties and stability. The iodophenyl analogue has been found to bind to bacterioopsin rapidly. The pigment obtained from this analogue showed a dramatically altered opsin shift of 1343 cm^-1. The anthryl analogue based bacteriorhodopsin, however, showed an opsin shift of 3849 cm^-1. It has been found that bacteriorhodopsin is quite unrestrictive in the ionone ring site. The apoprotein seems to prefer chromophores that have the ring portion co-planar with the polyene side chain. The purple membrane has also been modified by treatment with fluorescamine, a surface active reagent specific for amino groups. Reaction under controlled stoichiometric conditions resulted in the formation of a modified pigment. The new pigment showed a band at 390 nm—indicative of fluorescamine reaction with amino group (s) of apoprotein-besides retaining its original absorption band at 560 nm. Analysis of the fluorescamine modified bacteriorhodopsin resulted in the identification of lysine 129 as the modified amino acid residue. Fluorescamine-modified-bacteriorhodopsin suspension did not release protons under photolytic conditions. However, proteoliposomes of fluorescamine-modified-bacteriorhodopsin were found to show proton uptake, though at a reduced rate. Presented at the 3rd National Symposium on Bioorganic Chemistry, 1987, Hyderabad.     

Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin

A water-soluble yellow protein, previously discovered in the purple photosynthetic bacterium Ectothiorhodospira halophila, contains a chromophore which has an absorbance maximum at 446 nm. The protein is now shown to be photoactive. A pulse of 445-nm laser light caused the 446-nm peak to be partially bleached and red-shifted in a time less than 1 microsecond. The intermediate thus formed was subsequently further bleached in the dark in a biphasic process occurring in approximately 20 ms. Finally, the absorbance of native protein was restored in a first-order process occurring over several seconds. These kinetic processes are remarkably similar to those of sensory rhodopsin from Halobacterium, and to a lesser extent bacteriorhodopsin and halorhodopsin; although these proteins are membrane-bound, they have absorbance maxima at about 570 nm, and they cycle more rapidly. In attempts to remove the chromophore for identification, it was found that a variety of methods of denaturation of the protein caused transient or permanent conversion to a form which has an absorbance maximum near 340 nm. Thus, by analogy to the rhodopsins, the absorption at 446 nm in the native protein appears to result from a 106-nm red shift of the chromophore induced by the protein. Acid denaturation followed by extraction with organic solvents established that the chromophore could be removed from the protein. It is not identical with all-trans-retinal and remains to be identified, although it could still be a related pigment. The E. halophila yellow protein has a circular dichroism spectrum which indicates little alpha-helical secondary structure (19%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H⁺ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [³H]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of d -glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-β-d -glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni²⁺-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.     

Synthesis and Properties of Bacteriorhodopsin Analogs Containing Electron-Density Labels in the Chromophore Moiety

A method for synthesis of retinal analogs labeled with electron-density groups is suggested. The interaction of these polyene compounds with bacterioopsin in apomembrane of Halobacterium salinarum was tested. A retinal analog containing a crown-ether receptor group is able to interact readily with bacterioopsin giving rise to rapid formation of a pigment with absorption maximum at 460 nm. This pigment is capable of undergoing cyclic photoconversion. The crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency is very low (3% of that in native bacteriorhodopsin). This photocycle includes an M-like intermediate with a differential absorption maximum at 380 nm. A retinal analog in which the -ionone ring is replaced by ferrocene moiety forms a stable chromoprotein with the main absorption band at 483 nm and a shoulder near 590-610 nm.     

Femtosecond nuclear oscillations under charge separation in reaction centers of photosynthesis

Results are presented of a study of primary processes of formation of the charge separated states P⁺B_A ^- and P⁺H_A ^- (where P is the primary electron donor, B_A and H_A the primary and secondary electron acceptors) in native and pheophytin-modified reaction centers (RCs) of Rhodobacter sphaeroides R-26 by methods of femtosecond spectroscopy of absorption changes at low temperature. Coherent oscillations were studied in the kinetics at 935 nm (P* stimulated emission band), at 1020 nm (B_A ^- absorption band), and at 760 nm (H_A absorption band). It was found that when the wavepacket created under femtosecond light excitation approaches the intersection between P* and P⁺B_A ^- potential surfaces at 120- and 380-fsec delays, the formation of two electron states emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A ^-) takes place. At the later time the wavepacket motion has a frequency of 32 cm^-1 and is accompanied by electron transfer from P* to B_A in pheophytin-modified and native RCs and further to H_A in native RCs. It was shown that electron transfer processes monitored by the 1020-nm absorption band development as well as by bleaching of 760-nm absorption band have the enhanced 32 cm^-1 mode in the Fourier transform spectra.     

Spectral, Photophysical, and Stability Properties of Isolated Photosystem II Reaction Center

Photosystem II reaction center (RC) preparations isolated from spinach (Spinacea oleracea) by the Nanba-Satoh procedure (O Nanba, K Satoh 1987 Proc Natl Acad Sci USA 84: 109-112) are quite labile, even at 4°C in the dark. Simple spectroscopic criteria were developed to characterize the native state of the material. Degradation of the RC results in (a) blue-shifting of the red-most absorption maximum, (b) a shift of the 77 K fluorescence maximum from ~682 nm to ~670 nm, and (c) a shift of fluorescence lifetime components from 1.3-4 nanoseconds and >25 nanoseconds to ~6-7 nanoseconds. Fluorescence properties at 77 K seem to be a more sensitive spectral indicator of the integrity of the material. The >25 nanosecond lifetime component is assigned to P680⁺ Pheophytin⁻ recombination luminescence, which suggests a correlation between the observed spectral shifts and the photochemical competence of the preparation. Substitution of lauryl maltoside for Triton X-100 immediately after RC isolation stabilizes the RCs and suggests that Triton may be responsible for the instability.     

Rhodopsin in nanodiscs has native membrane-like photointermediates

Time-dependent studies of membrane protein function are hindered by extensive light scattering that impedes application of fast optical absorbance methods. Detergent solubilization reduces light scattering but strongly perturbs rhodopsin activation kinetics. Nanodiscs may be a better alternative if they can be shown to be free from the serious kinetic perturbations associated with detergent solubilization. To resolve this, we monitored absorbance changes due to photointermediates formed on the microsecond to hundred millisecond time scale after excitation of bovine rhodopsin nanodiscs and compared them to photointermediates that form in hypotonically washed native membranes as well as to those that form in lauryl maltoside suspensions at 15 and 30 °C over a pH range from 6.5 to 8.7. Time-resolved difference spectra were collected from 300 to 700 nm at a series of time delays after photoexcitation and globally fit to a sum of time-decaying exponential terms, and the photointermediates present were determined from the spectral coefficients of the exponential terms. At the temperatures and pHs studied, photointermediates formed after photoexcitation of rhodopsin in nanodiscs are extremely similar to those that form in native membrane, in particular displaying the normal forward shift of the Meta I(480) ? Meta II equilibrium with increased temperature and reduced pH which occurs in native membrane but which is not observed in lauryl maltoside detergent suspensions. These results were obtained using the amount of rhodopsin in nanodiscs which is required for optical experiments with rhodopsin mutants. This work demonstrates that late, physiologically important rhodopsin photointermediates can be characterized in nanodiscs, which provide the superior optical properties of detergent without perturbing the activation sequence.     

Stabilization of partially folded states of cytochrome c in aqueous surfactant: effects of ionic and hydrophobic interactions

The interaction of submicellar concentrations of sodium dodecyl sulfate (SDS) with horse heart cytochrome c has been found to stabilize two spectroscopically distinct partially folded intermediates at pH 7. The first intermediate is formed by the interaction of SDS with native cytochrome c, and this intermediate retains the majority of the secondary structure while the tertiary structure of the protein is lost. The unfolding of this intermediate with urea leads to the formation of a second intermediate, which is also formed on refolding of the unfolded protein (unfolded by urea) by SDS. The second intermediate retains about 50% of the native secondary structure with no tertiary structure of the protein. The second intermediate was found to be absent at low pH. While induction of helical structure of a protein by SDS in the native condition has been reported earlier, this is possibly the first report of the refolding of a protein in a strongly denaturing condition (in the presence of 10 M urea). The relative contributions of the hydrophobic and the electrostatic interactions of the surfactants with cytochrome c have been determined from the formation of the molten globule species from the acid-induced unfolded protein in the presence of SDS or lauryl maltoside.     

Conversion of CuA to a type II copper in cytochrome c oxidase

When cytochrome c oxidase is incubated at 43 degrees C for approximately 75 min in a solution containing the zwitterionic detergent sulfobetaine 12, the CuA site is converted into a type II copper as judged by changes in the 830-nm absorption band and the EPR spectrum of the enzyme. SDS-PAGE and sucrose gradient ultracentrifugation indicate concomitant loss of subunit III and monomerization of the enzyme during the heat treatment. Comparison of the optical and resonance Raman spectra of the heat-treated and native protein shows that the heme chromophores are not significantly perturbed; the resonance Raman data indicate that the small heme perturbations observed are limited to the cytochrome a3 site. Proton pumping measurements, conducted on the modified enzyme reconstituted into phospholipid vesicles, indicate that these vesicles are unusually permeable toward protons during turnover, as previously reported for the p-(hydroxymercuri)benzoate-modified oxidase and the modified enzyme obtained by heat treatment in lauryl maltoside. The sulfobetaine 12 modified enzyme is no longer capable of undergoing the recently reported conformational transition in which the tryptophan fluorescence changes upon reduction of the low-potential metal centers. Control studies on the monomeric and subunit III dissociated enzymes suggest that the disruption of this conformational change in the heat-treated oxidase is most likely associated with perturbation of the CuA site. These results lend support to the suggestion that the fluorescence-monitored conformational change of the native enzyme is initiated by reduction of the CuA site [Copeland et al. (1987) Biochemistry 26, 7311].     

Time and concentration dependence of the dicyclohexylcarbodiimide inhibition of proton movements in the cytochromebc 1 complex from yeast mitochondria reconstituted into proteoliposomes

The cytochromebc ₁ complex was isolated from yeast mitochondria solubilized with the detergent dodecyl maltoside and reconstituted into proteoliposomes to measure electrogenic proton pumping. Optimal respiratory control ratios of 4.0, obtained after addition of the uncoupler CCCP, and H⁺/e ^– ratios of 1.6 were obtained when the proteoliposomes were prepared with egg yolk phosphatidylcholine supplemented with cardiolipin. Moreover, it was critical to remove excess dodecyl maltoside in the final concentrated preparation prior to reconstitution to prevent loss of enzymatic activity. The rate of electrogenic proton pumping, the respiratory control ratios, and the H⁺/e ^– ratios were decreased by incubation of the cytochromebc ₁ complex with dicyclohexylcarbodiimide (DCCD) in a time and concentration dependent manner. Maximum inhibitions were observed when 50 nmol DCCD per nmol of cytochromeb were incubated for 30 min at 12°C with the intact cytochromebc ₁ complex. Under these same conditions maximum labeling of cytochromeb with [¹⁴C] DCCD was reported in a previous study [Beattieet al. (1984).J. Biol. Chem. 259, 10562–10532] consistent with a role for cytochromeb in electrogenic proton movements.     

Excitation energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus: A specific effect of 1-hexanol on the optical properties of baseplate and energy transfer processes

The effect of 1-hexanol on spectral properties and the processes of energy transfer of the green gliding photosynthetic bacterium Chloroflexus aurantiacus was investigated with reference to the baseplate region. On addition of 1-hexanol to a cell suspension in a concentration of one-fourth saturation, a specific change in the baseplate region was induced: that is, a bleach of the 793-nm component, and an increase in absorption of the 813-nm component. This result was also confirmed by fluorescence spectra of whole cells and isolated chlorosomes. The processes of energy transfer were affected in the overall transfer efficiency but not kinetically, indicating that 1-hexanol suppressed the flux of energy flow from the baseplate to the B806-866 complexes in the cytoplasmic membranes. The fluorescence excitation spectrum suggests a specific site of interaction between bacteriochlorophyll (BChl) c with a maximum at 771 nm in the rod elements and BChl a with a maximum at 793 nm in the baseplate, which is a funnel for a fast transfer of energy to the B806-866 complexes in the membranes. The absorption spectrum of chlorosomes was resolved to components consistently on the basis, including circular dichroism and magnetic circular dichroism spectra; besides two major BChl c forms, bands corresponding to tetramer, dimer, and monomer were also discernible, which are supposed to be intermediary components for a higher order structure. A tentative model for the antenna system of C. aurantiacus is proposed.Abbreviations A670 a component whose absorption maximum is located at 670 nm - (B)Chl (bacterio)chlorophyll - CD circular dichroism - F675 a component whose emission maximum is located at 675 nm - FMO protein Fenna-Mathews-Olson protein - LD linear dichroism - LH light-harvesting - McD magnetic circular dichroism - PS photosystem - RC reaction center     

Sensory rhodopsin I: Receptor activation and signal relay

Recent progress is summarized on the mechanism of phototransduction by sensory rhodopsin I (SR-I), a phototaxis receptor inHalobacterium halobium. Two aspects are emphasized: (i)The coupling of retinal isomerization to protein conformational changes. Retinal analogs have been used to probe chromophore-apoprotein interactions during the receptor activation process. One of the most important results is the finding of a steric trigger deriving from the interaction of residues on the protein with a methyl group near the isomerizing bond of the retinal (at carbon 13). Recent work on molecular genetic methods to further probe structure/function includes the synthesis and expression of an SR-I apoprotein gene designed for residue replacements by cassette mutagenesis, and transformation of anH. halobium mutant lacking all retinylidene proteins known in this species to SR-I⁺ and bacteriorhodopsin (BR)⁺. (ii)The relay of the SR-I signal to a post-receptor component. A carboxylmethylated protein (MPP-I) associated with SR-I and found in theH. halobium membrane exhibits homology with the signaling domain of eubacterial chemotaxis transducers (e.g.,Escherichia coli Tar, Tsr, and Trg proteins), suggesting a model based on SR-I MPP-I signal relay.     

Construction and purification of His-tagged staphylococcal ArsB protein,an integral membrane protein that is involved in arsenical salt resistance

Bacterial resistance to arsenical salts encoded on plasmid pI258 occurs by active extrusion of toxic oxyanions from cells of Staphylococcus aureus. The operon encodes for three gene products: ArsR, ArsB and ArsC. The gene product of arsB is an integral membrane protein and it is sufficient to provide resistance to arsenite and antimonite. A poly His-ArsB fusion protein was generated to purify the staphylococcal ArsB protein. Cells containing the His-tagged arsB gene were resistant to arsenite and antimonite. The levels of resistance to these toxic oxyanions by the His-tagged construct were greater than the levels obtained with the wild type gene. These data would indicate that the His-tagged protein is functionally active. A new 36 kDa protein band was visualized on 10% SDS-polyacrylamide gel electrophoresis (PAGE), which was confirmed as the His-ArsB protein by immunodetection with polyclonal Hisantibodies. The His-ArsB fusion protein was purified by the use of metal-chelate affinity chromatography with a Ni⁺²-nitrilotriacetic acid column and size-exclusion chromatography suggests that the protein was a homodimer.     

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.     

Cation-induced conformational changes in tRNA molecules

The uv absorption spectra and melting profiles of an initially ion-free solution of E. coli unfractionated tRNA are significantly modified by the addition of either Na⁺, Mg²⁺, or Mn²⁺ or of other first-series transition-metal ions such as Ni²⁺, Co²⁺, and Zn²⁺. The main effect of the addition of all monovalent or divalent cations examined is an increase of the ordered and stacking stabilized tRNA structure, as revealed by a drop in the absorption near 260 nm, as well as in the 4-TU absorption region. Sharp differences have, however, been detected in the 290–305-nm range in the presence of the various ions studied. When transition-metal ions were added to a tRNA solution, an absorption peak appeared at 294 nm. This effect is interpreted as a perturbation of the electronic structure of the bases due to direct binding of metal ions to the bases. An analysis of the variation in the spectrum as a function of metal concentration and of the thermal melting reversibility in the presence of various metal ions supports the conclusion that while all ions investigated are involved in binding to the phosphate groups of tRNA, transition-metal ions are also able to bind directly to the bases.     

Functional Overexpression and Purification of a Codon Optimized Synthetic Glucarpidase (Carboxypeptidase G2) in Escherichia coli

Glucarpidase (former name: carboxypeptidase G2, or CPG2) is a bacterial enzyme that is widely used in detoxification of the cytotoxic drug, methotrexate, and in Antibody Directed Enzyme Prodrug Therapy for cancer treatment. The glucarpidase gene of Pseudomonas sp. strain RS-16 was previously cloned in E coli, but expresses at a level that is approximately 100-fold lower than in the native strain. In this study, a synthetic gene coding for glucarpidase was codon-optimised and synthesized for maximum expression in E. coli using the vector pET28a. Our work indicated that the enzyme was expressed to ~60% of the total host protein and that purification of the recombinant His-tagged protein could be achieved in a single step by Ni²⁺ charged column chromatography. The synthetic recombinant glucarpidase expressed within this system was biologically active and zinc dependant. Our study showed that Mg²⁺ as well as Mn²⁺ ions inhibit the activity of the recombinant enzyme.     

Specific binding of integrin alphaIIbbeta3 to RGD peptide immobilized on a nitrilotriacetic acid chip: a surface plasmon resonance study

Nitrilotriacetic acid has been routinely used in protein purification for its high affinity for His-tagged protein in the presence of Ni²⁺. Here we reported a type of nitrilotriacetic acid chip (NTA-chip) prepared by transferring NTA-DOGS containing a lipid monolayer to a 50 nm thick gold layer deposited on a glass slide. The surface binding ability of His-tagged protein and regeneration of NTA chip were characterized using a synthetic polypeptide P1 (His-His-His-His-His-His--aminohexanoic-Gly-Gly-Arg-Gly-Asp-Ser). The effect of divalent cations on integrin binding affinity for RGD ligand was investigated after P1 had been immobilized onto the sensor chip. The results show that the NTA-chip is a useful tool to immobilize His-tagged protein on the chip surface, and can provide a functional orientation for further investigation. The results also show that removing of Ca²⁺ bound on low affinity sites or adding of Mn²⁺ can increase the binding ability of integrin.     

Topology of Cytochrome C Oxidase-Containing Proteoliposomes: Probes,Proteins and PH Gradients

Abstract

Cytochrome c oxidase-containing proteoliposomes (COV) prepared by cosonication show random orientation (45:55 in:out) of incorporated oxidase molecules; dialysed COV show 30:70 (in:out). Prepared COV show a pH gradient with an internal pH typically more acid than the medium. Such passive pH gradients probably reflect a Donnan distribution of anions such as chloride. The fluorescent pH probe 4-heptadecyl-7-hydroxycoumarin (HDHC) distributes between the two lipid leaflets at a ratio of between 30:70 and 33:67 (in:out) in cosonicated COV as measured by acid/base responses and quenching by p-xylene-b/s-pyridinium bromide. The HDHC pK was 8.25 in lauryl maltoside micelles, but membrane-bound HDHC showed a continuum of values ranging from 8.25 to 10.5. Maximum fluorescence in alkali was greater in lauryl maltoside than in COV. Active ΔpH gradients (alkaline inside) were generated by reductant and cytochrome c with aerobic oxidase-containing proteoliposomes ± valinomycin and nigericin. The gradients exceed 1.0 pH unit at low fluxes, higher than with water-soluble probes. ΔpH maintained between the bulk phases far from the membrane may be less than that at the lipid/water interface. With valinomycin (ΔΨ = 0), which accelerates ΔpH formation, ΔpH saturates at 1.0–1.2 units. Almost all the ΔΨ across the membrane can be converted into ΔpH by slow cation movement in the absence of ionophores. A gradient of either -90 mV (ΔΨ) or 1.0 pH unit (ΔpH) diminishes oxidase turnover by 80–90%. Control exerted by thermodynamically equivalent gradients is more effective with ΔpH than with ΔΨ. Differences between COV and mitochondria may be due to different rate-limiting electron transfer steps in the two systems.