Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

Characterization of mitochondrial aspartate aminotransferase from the liver of pyridoxine-deficient rats

The properties of crude and purified mitochondrial aspartate aminotransferase preparations from pyridoxine-deficient and control rat livers were compared. The preparations from the two sources showed very similar behaviors on heat treatment, electrophoresis and chromatofocusing, and had similar molecular weights, but their visible absorption spectra and circular dichroism properties were different. These results suggest that mitochondrial aspartate aminotransferase from pyridoxine-deficient and control rat livers have very similar properties, but differ somewhat in conformation in the region of the pyridoxal phosphate binding site.     

Linear dichroism of chicken cytosol aspartate aminotransferase oriented in polyacrylamide gel

Cytosolic chicken heart aspartate aminotransferase (EC 2.6.1.1) was incorporated in polyacrylamide gel and partially oriented by compressing the gel block in two mutually perpendicular directions. The linear dichroism (LD) was recorded in a dichrograph equipped with a quarter-wavelength device which transforms circularly polarized light into linearly polarized. Spectra were resolved with lognormal distribution curves. A marked difference has been found between reduced linear dichroism values (LD/A) in the absorption bands of the protonated (430 nm) and nonprotonated (360 nm) forms of the internal pyridoxal phosphate--lysine aldimine. This finding indicates that protonation of the internal aldimine bond induces a change in direction of the transition dipole moment within the coenzyme ring or reorientation of the ring. Formation of the external aldimine with 2-methylaspartate is accompanied by a decrease of the reduced LD value in the 430 nm band. On the other hand, binding of the dicarboxylate anions, which imitates formation of the noncovalent adsorption Michaelis complex, results in a marked increase of the reduced LD value in the 430 nm band. These data suggest that the coenzyme ring tilts in opposite directions upon noncovalent substrate binding and upon subsequent formation of the external aldimine.     

Linear dichroism characteristics of ethidium-and proflavine-supercoiled DNA complexes

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electrophoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA-bound EB molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism

Linear dichroism (LD) can be used to study the alignment of absorbing chromophores within long molecules. In particular, Couette flow LD has been used to good effect in probing ligand binding to DNA and to fibrous proteins. This technique has been previously limited by large sample requirements. Here we report the design and application of a new micro-volume Couette flow cell that significantly enhances the potential applications of flow LD spectroscopy by reducing the sample requirements for flow linear dichroism to 25 microL (with concentrations such that the absorbance maximum of the sample in a 1-cm pathlength cuvette is approximately 1). The micro-volume Couette cell has also enabled the measurement of fluorescence-detected Couette flow linear dichroism. This new technique enables the orientation of fluorescent ligands to be probed even when their electronic transitions overlap with those of the macromolecule and conversely. The potential of flow-oriented fluorescence dichroism and application of the micro-volume Couette LD cell are illustrated by the collection of data for DNA with minor groove and intercalating ligands: DAPI, Hoechst, and ethidium bromide. As with conventional fluorescence, improved sensitivity compared with absorbance LD is to be expected after instrumentation optimization.     

Variation of transaminases and lactate dehydrogenase in irradiated Biomphalaria alexandrina snails

Effect of ultraviolet and gamma radiations on the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LD) in Biomphalaria alexandrina snails, the specific intermediate host of schistosomiasis, was investigated. Changes in the electrophoretic pattern of LD in the species under study were also taken as a measured parameter and the effect of gamma-irradiation on the glutathione content in the haemolymph of the snails have been included.     

Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin.

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.     

Light-induced reorientation in the purple membrane.

Reorientation of bacteriorhodopsin in the native purple membrane was studied by time-resolved linear dichroism spectroscopy (TRLD) over the millisecond time regime. The time responses observed in TRLD are distinctly different from the isotropic transient absorption (TA) at wavelengths in the range 550-590 nm, where the bacteriorhodopsin ground state absorbs. In contrast, the TA and TRLD responses have nearly identical time dependence at 410 and 690 nm, where the intermediates M and O, respectively, principally contribute. These results demonstrate ground-state bacteriorhodopsin reorientation triggered by the photocycle. The TRLD and TA data are analyzed to test models for reorientational motion. Rotational diffusion of ground-state bacteriorhodopsin cannot account for the details of the data. Rather, the results are shown to be consistent with a reversible reorientation of "spectator" (nonexcited) members of the bacteriorhodopsin trimer in the purple membrane in response to the photocycling member of the trimer. This response may be associated with cooperativity in the trimer.     

Circular dichroism study of bacteriorhodopsin-lipid interaction

Delipidated bacteriorhodopsin purified from purple membrane of H. halobium was reconstituted with the circular dichroism active phospholipid. The observed circular dichroism spectra in the 450-700 nm region characteristic of bacteriorhodopsin showed the temperature dependence characterized by a midpoint at ca. 45 degrees C and this spectral change showed the disaggregation of bacteriorhodopsin trimer to monomer. The circular dichroism spectra in the 250-400 nm region characteristic of the azo chromophore of phospholipid exhibited a remarkable temperature dependence synchronized with the disaggregation of bacteriorhodopsin, suggesting that a large proportion of the phospholipid is present as boundary lipid.     

pH dependence of bacteriorhodopsin thermal unfolding

The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.     

Orientation and linear dichroism of Mastigocladus laminosus phycocyanin trimer and Nostoc sp. phycocyanin dodecamer in stretched poly(vinyl alcohol) films

The linear dichroism (LD) spectra of the C-phycocyanin (C-PC) trimer disks oriented in poly(vinyl alcohol) films (PVA) at room temperature and at 95 K were determined. Utilizing the known atomic coordinates of the chromophores (Schirmer, T., Bode, W. and Huber, R. (1987) J. Mol. Biol. 196, 677-695) and theoretical estimates of the orientations of the transition dipole moments relative to the molecular framework, the LD spectra were simulated using the pairwise exciton interaction model of Sauer and Scheer (Biochim. Biophys. Acta 936 (1988) 157-170); in this model, the alpha 84 and beta 84 transition moments are coupled by an exciton mechanism, while the beta 155 chromophore remains uncoupled. Linear dichroism spectra calculated using this exciton model, as well as an uncoupled chromophore (molecular) model, were compared with experimental LD spectra. Satisfactory qualitative agreement can be obtained in both the exciton and molecular models using somewhat different relative values of the theoretically estimated magnitudes of the beta 155 oscillator strength. Because the relative contributions of each of the chromophores (and thus exciton components) to the overall absorption of the C-PC trimer are not known exactly, it is difficult to differentiate successfully between the molecular and exciton models at this time. The linear dichroism spectra of PC dodecamers derived from phycobilisomes of Nostoc sp. oriented in stretched PVA films closely resemble those of the C-PC trimers from Mastigocladus laminosus, suggesting that the phycocyanin chromophores are oriented in a similar manner in both cases, and that neither linker polypeptides nor the state of aggregation have a significant influence on these orientations and linear dichroism spectra. The LD spectra of oriented phycocyanins in stretched PVA films at low temperatures (95 K) appear to be of similar quality and magnitude as the LD spectra of single C-PC crystals (Schirmer, T. and Vincent, M.G. (1987) Biochim. Biophys. Acta 893, 379-385).     

Complete amino acid sequence of rat liver cytosolic alanine aminotransferase

The complete amino acid sequence of rat liver cytosolic alanine aminotransferase (EC 2.6.1.2) is presented. Two primary sets of overlapping fragments were obtained by cleavage of the pyridylethylated protein at methionyl and lysyl bonds with cyanogen bromide and Achromobacter protease I, respectively. The protein was found to be acetylated at the amino terminus and contained 495 amino acid residues. The molecular weight of the subunit was calculated to be 55,018 which was in good agreement with a molecular weight of 55,000 determined by SDS-PAGE and also indicated that the active enzyme with a molecular weight of 114,000 was a homodimer composed of two identical subunits. No highly homologous sequence was found in protein sequence databases except for a 20-residue sequence around the pyridoxal 5'-phosphate binding site of the pig heart enzyme [Tanase, S., Kojima, H., & Morino, Y. (1979) Biochemistry 18, 3002-3007], which was almost identical with that of residues 303-322 of the rat liver enzyme. In spite of rather low homology scores, rat alanine aminotransferase is clearly homologous to those of other aminotransferases from the same species, e.g., cytosolic tyrosine aminotransferase (24.7% identity), cytosolic aspartate aminotransferase (17.0%), and mitochondrial aspartate aminotransferase (16.0%). Most of the crucial amino acid residues hydrogen-bonding to pyridoxal 5'-phosphate identified in aspartate aminotransferase by X-ray crystallography are conserved in alanine aminotransferase. This suggests that the topology of secondary structures characteristic in the large domain of other alpha-aminotransferases with known tertiary structure may also be conserved in alanine aminotransferase.     

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.     

山莨菪碱对紫膜中菌紫质结构的影响及其与膜脂的关系

本文用吸收光谱和可见圆二色谱研究了不同浓度的山莨菪碱对紫膜中菌紫质结构的影响,并设计了用不同浓度的去垢剂Triton X-100作为脂环境的扰动剂,研究山莨菪碱对菌紫质的影响与膜脂关系的实验.结果表明山莨菪碱不仅影响菌紫质分子本身的构象变化而且扰动了菌紫质分子之间的激子偶联作用.通过吸收差光谱技术表明山莨菪碱对菌紫质结构的影响与膜脂密切相关并指出紫膜中菌紫质的三体结构对膜功能的贡献是不容忽视的.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

Selected enzymatic activities in fresh water snails, specific intermediate host for human schistosomiasis

The activities of aspartate aminotransferase (AST) (EC.2.6.1.1.) I, alanine aminotransferase (ALT) (EC.2.6.1.2) II and lactate dehydrogenase (LD) (EC.1.1.1.27) III have been measured in tissue homogenate and in haemolymph of Biomphalaria alexandrina snails, the specific intermediate host for the human parasitic disease schistosomiasis due to Schistosoma mansoni.     

Solid-state synthesis of cyclo LD-diphenylalanine: A chiral phase built from achiral subunits

The solid-state structure of LL/DD or LD/DL diphenylalanine diluted in KBr pellets is studied by infrared (IR) absorption and vibrational circular dichroism (VCD) spectroscopy. The structure depends on the absolute configuration of the residues. The natural LL diphenylalanine exists as a mixture of neutral and zwitterionic structures, depending on the humidity of the sample, while mostly the zwitterion is observed for LD diphenylalanine whatever the experimental conditions. The system undergoes spontaneous cyclization upon heating at 125°C, resulting to the formation of a diketopiperazine (DKP) dipeptide as the only product. The reaction is faster for LD than for LL diphenylalanine. As expected, LL and DD diphenylalanine react to form the LL and DD enantiomers of cyclo diphenylalanine. Interestingly, the DKP dipeptides formed from the LD or DL diphenylalanine show unexpected optical activity, with opposite VCD spectra for the products formed from the LD and DL reagents. This is explained in terms of chirality synchronization between the monomers within the crystal, which retain the symmetry of the reagent, resulting to the formation of a new chiral phase made from transiently chiral molecules.     

Role of internal water molecules in bacteriorhodopsin

Internal water molecules are considered to play a crucial role in the functional processes of proton pump proteins. They may participate in hydrogen-bonding networks inside proteins that constitute proton pathways. In addition, they could participate in the switch reaction by mediating an essential proton transfer at the active site. Nevertheless, little has been known about the structure and function of internal water molecules in such proteins. Recent progress in infrared spectroscopy and X-ray crystallography provided new information on water molecules inside bacteriorhodopsin, the light-driven proton pump. The accumulated knowledge on bacteriorhodopsin in the last decade of the 20th century will lead to a realistic picture of internal water molecules at work in the 21st century. In this review, I describe how the role of water molecules has been studied in bacteriorhodopsin, and what should be known about the role of water molecules in the future.     

Nonidentity of the Aspartate and the Aromatic Aminotransferase Components of Transaminase A in Escherichia coli

Tyrosine, added to the growth medium of a strain of Escherichia coli K-12 lacking transaminase B, repressed the tyrosine, phenylalanine, and tryptophan aminotransferase activities while leaving the aspartate aminotransferase activity unchanged. This suggested that the aspartate and the aromatic aminotransferase activities, previously believed to reside in the same protein, viz. transaminase A, are actually nonidentical. Further experiments showed that, upon incubation at 55 C, the aspartate aminotransferase of crude extracts was almost completely stable, whereas the tyrosine and phenylalanine activities were rapidly inactivated. Apoenzyme formation was faster, and apoenzyme degradation proceeded more slowly with aspartate aminotransferase than with tyrosine aminotransferase. Electrophoresis in polyacrylamide gels separated the aminotransferases. A more rapidly moving band contained tyrosine, phenylalanine, and tryptophan aminotransferases, and a slower band contained aspartate aminotransferase. A mutant of E. coli K-12 with low levels of aspartate aminotransferase exhibited unchanged levels of tyrosine aminotransferase. Thus, transaminase A appears to be made up of at least two proteins: one of broad specificity whose synthesis is repressed by tyrosine and another, specific for aspartate, which is not subject to repression by amino acids. The apparent molecular weights of both the aspartate and the aromatic aminotransferases, determined by gel filtration, were about 100,000.     

Steady-state and transient polarized absorption spectroscopy of photosystem I complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus

Core antenna and reaction centre of photosystem I (PS I) complexes from the cyanobacteria Arthrospira platensis and Thermosynechococcus elongatus have been characterized by steady-state polarized absorption spectroscopy, including linear dichroism (LD) and circular dichroism (CD). CD spectra and the second derivatives of measured 77 K CD spectra reveal the spectral components found in the polarized absorption spectra indicating the excitonic origin of the spectral forms of chlorophyll in the PS I complexes. The CD bands at 669-670(+), 673(+), 680(-), 683-685(-), 696-697(-), and 711(-) nm are a common feature of used PSI complexes. The 77 K CD spectra of the trimeric PS I complexes exhibit also low amplitude components around 736 nm for A. platensis and 720 nm for T. elongatus attributed to red-most chlorophylls. The LD measurements indicate that the transition dipole moments of the red-most states are oriented parallel to the membrane plane. The formation of P700(+)A(1)(-) or (3)P700 was monitored by time-resolved difference absorbance and LD spectroscopy to elucidate the spectral properties of the PS I reaction centre. The difference spectra give strong evidence for the delocalization of the excited singlet states in the reaction centre. Therefore, P700 cannot be considered as a dimer but should be regarded as a multimer of the six nearly equally coupled reaction centre chlorophylls in accordance with structure-based calculations. On the basis of the results presented in this work and earlier work in the literature it is concluded that the triplet state is localized most likely on P(A), whereas the cation is localized most likely on P(B).