TIME-RESOLVED FLUOROMETRY OF BACTERIORHODOPSIN

Abstract A mode-locked Nd:YAG laser was used to excite the aromatic amino acid residues of bacteriorhodopsin in the purple membrane and the tryptophan (Trp) fluorescence decay analyzed with a streak camera (λ> 380 nm). The decay kinetics are resolvable into two first-order half-times (1.5 and 0.17 ns, respectively), while for retinylidene-free bacterioopsin, only the longer-lived Trp emission was observed. The shorter-lived species reappeared upon regeneration of bacteriorhodopsin by addition of retinal to bacterioopsin but not on treatment of the latter with an equivalent of retinol. It is proposed that these results are consistent with a structural model in which the 7-8 Trp's distributed among sections A, C, E and F of the seven helical segments A-G of native bacteriorhodopsin are distinguishable by their distances from the chromophore. Assuming a Förster mechanism for energy transfer with R_o= 25 and 32 Å, respectively, for retinylidene chromophore and retinol the Trp's may be divided into two groups: (i) those completely quenched by retinol and partly quenched by retinal (τ= 0.17) with R ≃ 18 Å and (ii) those (τ= 1.5 ns) which are quenched neither by chromophore nor retinol with R > ca. 30 Å. These results are consistent with and support some of the best models of Engelman et al. (1980) for the protein conformation in the purple membrane.     

INACTIVATTON OF S-RNA BY ULTRAVIOLET RADIATIONS

Abstract. Various S-RNA's from E. coli show wide differences in sensitivity to ultraviolet (u.v.) radiation as measured by their amino acid acceptor capacities. S-RNA for valine is least sensitive and phenylalanine and lysine are most sensitive. S-RNA's for valine, isoleucine, glycine, aspartic acid, serine, and arginine give exponential type dose-effect curves. Those for alanine, glutamic acid, lysine, and phenylalanine are rapidly inactivated at low doses and only at higher doses give exponential type inactivation curves having lower slopes than seen at low doses. Penylalanine and glutamic acid S-RNA inactivated by 2650 or 2804 Å radiations are not reactivated by 2380 Å radiation, indicating that uracil dimers are not involved in the inactivation process. Heating of irradiated S-RNA to bring about rehydration of uracil residues is not effective in restoring acceptor activity of phenylalanine and leucine S-RNA. Melting curve experiments suggest that photochemical products other than uracil diniers and hydrated uracil residues are formed and result in changes in the secondary structure of S-RNA.     

INTERACTIONS OF BOTH MELITTIN AND ITS SITE-SPECIFIC MUTANTS WITH BACTERIORHODOPSIN OF Halobacterium halobium: SITES OF ELECI'ROSTATIC INTERACI'ION ON MELITI'IN

Abstract Melittin and its site-specific mutants differentially delay the slow-decaying component of the photocycle intermediate M₄₁₂ of bacteriorhodopsin in the purple membrane and the acetylated purple membrane whose several lysine residues are modified. This effect is attributed to the interaction of the total positive charges of melittin or its mutants with the total negative charges of bacteriorhodopsin. The effects of melittin and its mutants on the Triton X-100–solubilized bacteriorhodopsin monomers are somewhat complicated but are associated with their charges. These results show that there is electrostatic interaction between bacteriorhodopsin and melittin and that both N-and C-termini of melittin function as sites of the interaction, with Arg 22 and Arg 24 making a prominent contribution to the effective surface charge of melittin. Melittin, at certain concentrations, partially restores the decreased photoactivity of the bacteriorhodopsin monomers trapped in the Triton-lipid-protein mixed micelles, which suggests that melittin may compete with Triton X-100 for the binding sites on the bacteriorhodopsin monomers. Other kinds of interactions between bacteriorhodopsin and melittin are also indicated. The possible states of melittin in membranes are discussed.     

P588, A SECOND RETINAL-CONTAINING PIGMENT IN HALOBACTERIUM HALOBIUM

Abstract— Halobacterium mutant strains with defects in the biosynthesis of various pigments have been isolated. One of these strains, mutant ET-15, is incapable of producing the light-driven proton pump bacteriorhodopsin and the carotenoid bacterioruberin. However, ET-15 synthesizes another photochemically active, retinal-containing pigment, P₅₈₈, which mediates light-induced proton uptake enhanced by uncouplers. P₅₈₈ and bacteriorhodopsin are simultaneously present in wild-type cells grown under normal conditions; however, they can be distinguished by the following criteria.

• 1 They can be separated by independent mutational events.
• 2 Proton ionophores such as FCCP diminish bacteriorhodopsin-driven proton translocation but enhance P₅₈₈-mediated proton flows. We define here proton translocations which can be diminished by the addition of uncouplers (e.g. FCCP) as ‘active’, others which can be enhanced by FCCP as ‘passive’.
• 3 The kinetic and spectral properties of the photocycle intermediates of bacteriorhodopsin and P₅₈₈ are different.
• 4 The action spectrum for photocycling of P₅₈₈ is red-shifted with respect to that for bacteriorhodopsin.

A comparison of action spectra for proton translocations with that for photocycling links pigment P₅₈₈ and light-driven, passive proton uptake by ET-15 envelope vesicles. When chemically bleached P₅₈₈ was regenerated with all-trans-retinal, both photochemical activity and light-induced proton inflow were restored to equal extents. This identifies P₅₈₈ with the energizer for the passive proton flows.     

ALKALINE QUENCHING OF BACTERIORHODOPSIN TRYPTOPHANYL FLUORESCENCE: EVIDENCE FOR AQUEOUS ACCESSIBILITY OR A HYDROGEN-BONDED CHAIN

Abstract— Comparison between Trp fluorescence yields of membrane-bound bacteriorhodopsin (BR) and retinylidene-free bacterioopsin (BO) is consistent with a model in which all eight Trp residues are active fiuorophores in the latter, while the emission of all but two residues in the former is lost by energy transfer to retinal. The visible chromophore of BR is progressively bleached with increasing pH. Up to pH 12 this bleaching is reversed on reneutralization; but above this the change is irreversible with the appearance of a new absorption band characteristic of free retinal. Emission yields for both proteins decrease with increasingly alkaline pH in a manner typical of energy transfer to weakly-fluorescent tyrosinate. The limiting yields, reached at a pH corresponding to that producing irreversible bleaching of the visible chromophore, agree with an integral value of one remaining active Trp fluorophore in BR and between one and two in BO and show that the bulk of Trp are within the 11 Å Förster energy-transfer distance of Tyr accessible to OH⁻. Current models of the native protein structure of BR arrange the polypeptide chain primarily in a bundle of seven helical segments with axes perpendicular to the lipid bilayer plane and with buried polar residues, including Trp and Tyr, located at intrahelical surfaces. An interpretation of the observed accessibility of buried Tyr to OH⁻ is that an aqueous region exists within the protein structure. Moreover, this putative aqueous region must be close to the retinylidene chromophore and thus may be associated with the light-driven ion transport system. The results are also compatible with energy transfer to internal Tyr residues which are connected via a chain of phenolate hydrogen bonds to a surface Tyr.     

Chromatographic Analysis of Suberimidate-Crosslinked Lysine

Abstract

Quantitative analysis of bislysylsuberamidine and monolysylsuberamidinic acid, which are obtained by an acid hydrolysis of protein cross-linked with dimethyl suberimidate, on an amino acid analyzer are described. Both of ninhydrin and fluorometric detection with o-phthalaldehyde were applied and less than 50 pmol of cross-linked lysine was analyzed in the latter case. The first-order rate constant for hydrolysis of amidine bond under standard conditions of acid hydrolysis of protein was found to be 3.4 × 10^?3h^?1.     

High Resolution Structure of Bacteriorhodopsin Determined by Electron Crystallography

Abstract— Bacteriorhodopsin pumps protons from the cytoplasm to the outside of halobacteria, Halobacterium salinarium , by using absorbed light energy. The newly observed density map at 3 Å resolution clarified nearly the entire structure; the resolution in the direction perpendicular to the membrane surface is 3.2 Å. The new structure clearly indicates the proton transfer pathway in bacteriorhodopsin. In particular, the location of key aspartic acid and glutamic acid residues in the derived structural model suggested funneling structures with different designs for input and output of protons on the cytoplasmic and extracellular sides, respectively, of the protein. This paper describes the major differences between the model based on the new observation and the former model obtained through crystallographic refinement by Grigorieff et al . ( J. Mol. Biol 259; 393-421, 1996).     

Influence of bifurcation position and length of side chains on the structure of isoindigo-based conjugated polymer thin films

We chose a series of isoindigo-based conjugated polymer(ⅡDDT,ⅡDDT-C3 and ⅡDDT-C4) with different length of side chains and bifurcation positions to investigate the relationship between the degree of alignment and the length of side chains and bifurcation positions.We found that the dichroic ratio was increased from 2.37 to 5.23 when the side chain was longer and the bifurcation position was away from the backbone.The π-π stacking distance was decreased from 3.67 A to 3.61 A when the bifurcation position was away from the backbone because of its smaller hindrance and the d-spacing of the(100)was increased from 20.06 A to 25.21 A when the side chain was longer.All the polymers were adopted an edge-on orientation with the backbone paralleled with the long axis of fibers.The weak interaction of side-chain in ⅡDDT-C4 was beneficial for the molecules being rearranged in parallel during the contact line receding and the strong n-n interaction could accelerate the interchain assembly of the parallel molecules through π-π interaction to form aligned fibers.     

Grease immobilized on polyethyleneimine cotton cloth

Jack bean urease was immobilized on polyethylenimine-coated cotton cloth by adsorption following by crosslinking with dimethyl suberimidate. Of the various methods used, crosslinking with dimethyl suberimidate was found to stabilize the enzyme with minimal inactivation. Cloth-bound urease showed a shift in pH optimum towards the acidic side without appreciable change in temperature optimum and thermostability. Cloth-bound urease could be used repeatedly for urea hydrolysis without appreciable loss in activity. Alternatively, urease cloth could be stitched in the form of a bag containing ammonia adsorbent and used for urea hydrolysis with simultaneous removal of ammonia.     

A crosslinked preparation ofE. coli β-D-galactosidase

β-D-Galactosidase fromE. Coli was crosslinked using glutaraldehyde and two bisimidoesters. With glutaraldehyde and dimethyl adipimidate (DMA), it is possible to obtain preparations having higher activity than the native enzyme. Glutaraldehyde and DMA gave preparations showing enhanced thermal stability. The preparation crosslinked with DMA, when used for continuous hydrolysis of lactose in milk, was found to be significantly better than the native enzyme.     

THE ATTACHMENTS OF PHYCOBILINS TO CYSTEINYL RESIDUES

The attachments of phycobilins to cysteinyl residues have been worked out through the reactions of phycoerytbrobilin dimethyl ester and phycocyanobilin dimethyl ester with cysteine methyl ester and reduced glutathione dimethyl ester respectively. A series of model compounds which carry the same conjugative skeletons as the chromophores in phycobillproteins have been purified anti identified. These compounds are characterized with strong fluorescence emission and circular dichroism effects which appeared weaker for the common bilinoid chromophores. Analyses of the circular dichroism effects of these compounds indicated that the major reaction products of phycobilins with cysteine methyl ester and reduced glutathione dimethyl ester carried the same stereochemical configuration as the chromophores in phvcobilinoroteins     

KINETICS OF DIMER FORMATION AND PHOTOHYDRATION IN ULTRAVIOLET-IRRADIATED POLYURIDYLIC ACID

Abstract— The decrease in absorbance at 2600 Å of poly-U following u.v. irradiation is analyzed quantitatively in terms of uracil-dimer formation and photohydration of uracil residues. At all wave lengths tested between 2300 and 2800 Å both dirner formation and hydration occur as well as dimer breakage. The cross section for uracil dirner breakage is large at 2300 and decreases to a relatively small value at 2800 Å. Consequently, at the shorter wave lengths the steady state for dimer formation and breakage is reached at lower doses than for the longer wave lengths. Absorbance decreases caused by longer wave lengths can be partially restored by irradiation with 2380 Å. At high doses, for all wave lengths, the main photoproducts are hydrated uracil residues. The maximum number of dimers that may be formed increases with wavelength. The absorbance loss caused by photohydration may be reversed by acidification and heat. For 2650 Å irradiation, 95 per cent of the absorbance is restored by a combination of the 2380 Å and thermal treatment. The values for the photochemical reactions of poly-U are fairly close to those for poly-T and for uridine.     

PHOTOSELECTION AND TRANSIENT LINEAR DICHROISM WITH ORIENTED IMMOBILIZED PURPLE MEMBRANES: EVIDENCE FOR MOTION OF THE C(20)-METHYL GROUP OF THE CHROMOPHORE TOWARDS THE CYTOPLASMIC SIDE OF THE MEMBRANE*

Abstract— We have determined the transition dipole moment orientation of the chromophore during the photocycle of bacteriorhodopsin by photoselection and time-resolved linear dichroism experiments with samples of oriented immobilized purple membranes. This technique offers two important advantages over experiments with isotropic aqueous suspensions: (1) the depolarization due to the rotational diffusion of the membranes is eliminated, (2) the sensitivity for detecting the orientation of the transition dipole moment of intermediates is greatly increased. The appropriate equations for the analysis of time-resolved linear dichroism experiments with samples of oriented immobilized membranes will be presented. In the transition from the ground state of bacteriorhodopsin to the M-intermediate, the transition dipole moment tilts out of the plane of the membrane by about 3°. On the basis of current structural information on the plane of the chromophore and the orientation of its C(19) and C(20) methyl groups, a tilt of the transition dipole moment into the plane of the membrane would have been expected if it is assumed that the orientation of the conjugated polyene chain from C(5) to C(13) is the same in both states. The experimental result may be explained by an 11° tilt of the C(5) to C(13) part of the chain out of the plane of the membrane with the C(20) methyl group moving towards the cytoplasmic side of the membrane by about 1.7 Å and the cyclohexene ring staying fixed. This interpretation is supported by recent neutron diffraction experiments on the chromophore position in the M-intermediate.     

[MESITYL]BACTERIORHODOPSIN. THE PROPERTIES OF AN ANALOGUE OF THE PURPLE MEMBRANE CONTAINING [MESITYL] RETINAL AS THE CHROMOPHORE

Abstract— A new synthesis of all-trans-[mesityl]retinal, II , (all-trans-3,7-dimethyl-9-(2',4',6'-trimethylphenyl)-2,4,6,8,-nonatetraenal) and 13-cis-[mesityl]retinal, VI , (3,7-dimethyl-9-(2'4'6'-trimethylphenyl)-2Z,4E,6E,8E-nonatetraenal) is reported. Combination of all-trans-[mesityl]retinal with bacterioopsin results in the formation of a synthetic membrane (Λ_max 460) which has photocycling properties similar to the purple membrane although its cycling rate is very much slower. An M-type intermediate can be trapped at -60°C. Photoreversal of the M-intermediate to the wavelength of initial absorption is observed. Phototransformation of the initial [mesityl]bacteriorhodopsin is accompanied by conversion of the all-frans to the 13- cis -isomer.     

Purification and some properties of tryptophan-5-monooxygenase from rabbit hindbrain

An affinity chromatography procedure for the rapid purification of tryptophan-5-monooxygenase from rabbit hindbrains was developed using e-aminocaproyl-D-tryptophan methyl ester-Sepharose-4B gels. The precise requirements for the optimal biospecific interaction between the affinity ligand and the ligate (enzyme) was established from a study of the effects of the variation in the length of the "spacer’’ on the affinity properties of the gel. The enzyme preparation isolated by this procedure was found to be essentially homogeneous and was characterized by a molecular weight of 200,000 ±20,000. SDS-polyacrylamide gel electrophoresis of the enzyme revealed it to be a dimer, the molecular weight of each subunit being approximately 90,000. The specific activity of the enzyme preparation is approxi-mately 7-10 times that of the crude homogenate, but a further fivefold enhancement in the specific activity could be obtained by limited proteolysis with trypsin. The extreme lability of the enzyme could be circumvented by its immobilization on activated Sepharose or by cross-linking with dimethyl suberimidate. The kinetic properties as well as the advantages of such stabilized enzyme preparations are presented.     

PROPERTIES OF SYNTHETIC BACTERIORHODOPSIN PIGMENTS. FURTHER PROBES OF THE CHROMOPHORE BINDING SITE

Abstract— A series of retinals with specific structural alterations have been synthesized to probe the bacteriorhodopsin binding site. The 4-chloro-, 4-bromo- and 4-iodoretinals all form pigments with bacterioopsin but undergo an in situ displacement of the allylic halogen to form the 4-hydroxyretinal pigment. Several naphthyl retinals were prepared which effectively extend the polyene chain and/or add bulk to the ring portion of the chromophore. All the naphthyl retinals form pigments with bacterioopsin but only the pigment containing the derivative with a polyene side chain identical to that of retinal pumps protons efficiently. The 12-butyl-13-desmethylretinal was also synthesized but this analogue did not form a pigment with bacterioopsin. These results confirm the nonspecificity at the ring portion of the chromophore binding site and the importance of the role of the polyene chain in the proton pumping function of bacteriorhodopsin.     

THE ROLE OF ARGININES 82 AND 227 IN THE BACTERIORHODOPSIN PROTON PUMP

Abstract
Arginine residues 82 and 227 in bacteriorhodopsin were replaced by glutamine residues, using the site-directed mutagenesis techniques. Mutant bacteriorhodopsins were found to be competent in formation and decomposition of the photocycle M412 intermediate as well as in generation of photoelectric potential provided that pH of the medium is sufficiently high. Lowering of pH results in transition of bacteriorhodopsin into a blue acidic form which cannot produce M412 and photo-potential. The p K values of these transitions for Arg-227 → Gln and Arg-82 → Gln mutants are shifted correspondently for 1 and 4 pH units to a higher pH region in comparison with native bacteriorhodopsin. The rate of the M412 formation in both mutants was similar to that in the native protein. As to M412 decay, it is much slower in Arg-227 → Gln mutant than in native and Arg-82 → Gln bacteriorhodopsins. In all cases, the decay depends only slightly upon pH. It is concluded that Arg-82 is involved in maintenance of a bacteriorhodopsin structure that is resistant to the pH decrease down to 4 whereas Arg-227 is required first of all for the process of Schiff base reprotonation.     

Effects of chain length and N-methylation on a cation-pi interaction in a beta-hairpin peptide

The effects of N-methylation and chain length on a cation-pi interaction have been investigated within the context of a beta-hairpin peptide. Significant enhancement of the interaction and structural stabilization of the hairpin have been observed upon Lys methylation. Thermodynamic analysis indicates an increased entropic driving force for folding upon methylation of Lys residues. Comparison of lysine to analogues ornithine (Orn) and diaminobutyric acid (Dab) indicates that lysine provides the strongest cation-pi interaction and also provides the most stable beta-hairpin due to a combination of side chain-side chain interactions and beta-sheet propensities. These studies have significance for the recognition of methylated lysine in histone proteins.     

Effect of Substituents on the Homopolymerization Activity of Methyl Alkyl Diallyl Ammonium Chloride

Among nitrogen-containing cationic electrolytes, diallyl quaternary ammonium salt is a typical monomer with the highest positive charge density, which has attracted the most attention, especially in the research on homopolymers and copolymers of dimethyl diallyl ammonium chloride (DMDAAC), which occupy a very unique and important position. In order to improve the lipophilicity of substituted diallyl ammonium chloride monomers under the premise of high cationic charge density, the simplest, most direct, and most efficient structure design strategy was selected in this paper. Only one of the substituents on DMDAAC quaternary ammonium nitrogen was modified by alkyl; the substituents were propyl and amyl groups, and their corresponding monomers were methyl propyl diallyl ammonium chloride (MPDAAC) and methyl amyl diallyl ammonium chloride (MADAAC), respectively. The effect of substituent structure on the homopolymerization activity of methyl alkyl diallyl ammonium chloride was illustrated by quantum chemical calculation and homopolymerization rate determination experiments via ammonium persulfate (APS) as the initiator system. The results of quantum chemistry simulation showed that, with the finite increase in substituted alkyl chain length, the numerical values of the bond length and the charge distribution of methyl alkyl diallyl ammonium chloride monomer changed little, with the activation energy of the reactions in the following order: DMDAAC < MPDAAC < MADAAC. The polymerization activities measured by the dilatometer method were in the order DMDAAC > MPDAAC > MADAAC. The activation energies E_a of homopolymerization were 96.70 kJ/mol, 97.25 kJ/mol, and 100.23 kJ/mol, and the rate equation of homopolymerization of each monomer was obtained. After analyzing and comparing these results, it could be easily found that the electronic effect of substituent was not obvious, whereas the effect of the steric hindrance was dominant. The above studies have laid a good foundation for an understanding of the polymerization activity of methyl alkyl diallyl ammonium chloride monomers and the possibility of preparation and application of these polymers with high molecular weight.     

PHOTOOXIDATION OF LANTHANIDE ION-LYSOZYME COMPLEXES. A NEW APPROACH TO THE EVALUATION OF INTRAMOLECULAR DISTANCES IN PROTEINS

Abstract— Rare-earth metal ions give 1:1 complexes with hen's egg-white lysozyme. Spectroscopic and enzymic activity measurements suggest that the binding site consists of the side chains of glutamic-35 and aspartic-52. The spatial conformation of these complexes is practically identical to that of native lysozyme, especially as concerns the environment of the tryptophyl side chains. Irradiation of La³⁺-lysozyme by visible light, in the presence of proflavine as photosensitizer, causes the oxidative modification of all the tryptophyl and methionyl residues at almost the same rate as in uncomplexed protein. On the other hand, when lanthanide ions with nonvanishing magnetic moments were coordinated with lysozyme, at least some tryptophans and methionines were protected from photooxidative attack. The distance of the protected residues from the coordination site increased with increasing magnetic moment of the bound metal ion, which suggests that inhibition of the photoprocess was mainly due to perturbation of the lifetime of the electronically excited intermediate species. On the basis of the atomic coordinates of lysozyme in the crystal state, it is thus possible to define a "quenching radius" for the various lanthanide ions; these radii could in turn be used to evaluate intramolecular distances in proteins of unknown tertiary structure, by identifying the amino acid residues that are protected or photooxidized upon irradiation of complexes between the given protein and several different lanthanide ions. Our studieson lysozyme allow us to define five radii of protection, ranging from 6·7 Å for Sm³⁺ to over 17 Å for Dy³⁺, Ho³⁺, Er³⁺ and Tb³⁺. Therefore, this technique opens the possibility of mapping appreciably large regions of a protein molecule.