Comparative studies on the fine structure of purple membrane fromHalobacterium cutirubrum andHalobacterium halobium

Summary Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane fromHalobacterium cutirubrum andHalobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane fromHalobacterium halobium, it must be similarly so for the membrane fromHalobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.     

Effects of Bleaching and Regeneration on the Purple Membrane Structure of Halobacterium halobium

Sequential bleaching in the presence of hydroxylamine and subsequent regeneration of the purple membrane of Halobacterium halobium was studied by concomitant monitoring of its absorption and circular dichroic spectra in order to ascertain its effects on protein interaction(s) (which may result in possible excitonic interaction between the retinal chromophores), chromophore-apoprotein interaction(s), and protein conformational stability in the membrane. It was concluded that (a) although experimental results are consistent with an exciton mechanism for the interaction between retinal π - π* (NV₁) transition movements in the purple membrane, no evidence for such a mechanism for interaction between retinaloxime transition moments is apparent in the case of the bleached membrane; (b) the bacteriorhodopsin molecules organized in clusters of three in the membrane appear to bleach simultaneously; (c) the retinaloxime produced on bleaching the purple membrane in the presence of hydroxylamine is strongly optically active, because of dissymmetry-inducing and/or -selecting constraints on the chromophore by a component of the membrane (most likely the apoprotein), and when the membrane is regenerated by the addition of retinal, these constraints are lost; and (d) evidence from ultraviolet absorption and circular dichroic spectra suggests that the membrane apoprotein undergoes appreciable conformational changes involving tertiary structure on bleaching with no significant secondary structure involvement. These results are compared with recently reported results from this laboratory on the effects of bleaching on the bovine rod outer segment disk membrane structure.     

Orientations of the retinyl and the heme chromophores in the brown membrane of Halobacterium halobium

The orientations of the retinyl and heme chromophores of bacteriorhodopsin and cytochrome b-561 of the brown membrane of Halobacterium halobium have been determined by linear dichroic spectroscopy of oriented brown membrane films. Both chromophores exhibit cylindrical symmetry with respect to the membrane normal. However, the retinyl transition dipole moment is polarized at an angle of 20 to 24 ° with respect to the plane of the membrane while the plane of the heme is oriented nearly perpendicular to the membrane plane. Therefore, the orientation of retinal bound to bacterio-opsin in the brown membrane is approximately the same as in the purple membrane. This is supportive of our previous conclusions that the fine structures of the bacteriorhodopsins of these membranes are very similar in spite of differences in the composition and structure of the two membranes. The orientation of the heme plane of the membrane-bound cytochrome b-561 is very similar to orientations of several membrane-bound heme proteins that are involved in electron transfer processes and may be suggestive of its function in the brown membrane. Analysis of the linear dichroic spectrum over the entire bacteriorhodopsin band using an exciton formalism is in accord with the energy separation of the in-plane and out-of-plane excitonic transitions being less than 5 nm. Since a similar energy separation was reported for the purple membrane, the relative positions of the retinals must be approximately the same in both membranes. A similar analysis of the Soret region, based on the existence of two degenerate mutually perpendicular porphyrin transitions, indicates that the energy separation should be from 5 to 20 nm. However, the smaller value is unlikely for it would imply very large circular dichroic bands not yet encountered in any heme proteins.     

The Photocycle of Bacteriorhodopsin in the Two-Dimensional Orthorombic Crystal Form of Purple Membrane

Laser flash photolysis and low-temperature absorption studies of the photocycle of orthorhombic purple membrane (o-PM) reveal the existence of the same K, L, and M intermediates as found in the native hexagonal purple membrane (h-PM). However, the 0 intermediate is missing in the o-PM. The absorption spectrum of the K intermediate of o-PM is blueshifted by ~15 nm relative to the K intermediate found in the hexagonal purple membrane. The decay relaxation time constants of M in the o-PM are higher by more than an order of magnitude than the corresponding relaxation time constants in the h-PM. Similarly to the h-PM, the decay of M depends on the pulse width of excitation. The time-independent anisotropy factor obtained in photoselection studies of the M intermediate demonstrates the complete immobility of bacteriorhodopsin (bR) within the o-PM matrix. The same anisotropy factor of 0.3 obtained for o-PM and for h-PM suggests that in both crystalline lattices the transition moment of the retinal chromophore has similar angles with the plane of the membrane. The dependence of the decay kinetics of M on its occupancy may suggest the existence of kinetic coupling between neighboring bR molecules.     

Photoselection and circular dichroism in the purple membrane.

The transient dichroic ratio D = delta A parallel/delta A perpendicular has been measured in the visible absorption region of bacteriorhodopsin in purple membrane by a flash photolysis method. D is found to be wavelength independent throughout the visible absorption band, and reaches a maximum value of 2.75 +/- 0.15 on reduction of the excitation intensity. This value is close to that expected for a single nondegenerate transition dipole moment and is incompatible with the strong exciton coupling model used to explain circular dichroism (CD) spectrum of purple membrane. A time-dependent analysis of the exciton interaction and consideration of the coupling strength suggests an explanation of these observations. It is concluded that excitation interaction between retinals in purple membrane is of the weak or very weak type defined by Förster.     

Phase transitions of the purple membrane and the brown holo-membrane X-ray diffraction,circular dichroism spectrum and absorption spectrum studies

The phase transition of the purple membrane observed by differential scanning calorimetry (Jackson, M.B. and Sturtevant, J.M. (1978) Biochemistry 17, 911–915) has been investigated by X-ray diffraction, circular dichroism and absorption spectrum, in comparison with the phase transition in the brown holo-membrane. The two-dimensional crystal of bacteriorhodopsin transformed into two-dimensional liquid around 74–78°C in the purple membrane and around 50–60°C in the brown holo-membrane. The X-ray diffraction patterns obtained at 78°C for the purple membrane and at 60°C for the brown holo-membrane exhibit several broad peaks. Analysis of the pattern suggests that bacteriorhodopsin molecules aggregate in trimers even above the phase transition temperature. The negative circular dichroism band in the visible region is still present at 80°C in the purple membrane and at 60°C in the brown holo-membrane, but becomes negligibly small at 70°C in the brown holo-membrane. The 560 nm absorption peak due to bacteriorhodopsin changes its position and height drastically around 80°C in the brown holo-membrane as in the purple membrane. X-ray diffraction studies have been made on membranes of total lipids extracted from the purple membrane. No indication of the phase transition has been found between ?81°C and 77°C.     

The secondary structure of human plasma fibronectin: conformational changes induced by acidic pH and elevated temperatures; a circular dichroic study

The secondary structure of native human plasma fibronectin, based on circular dichroic spectra, has been estimated to contain 79% beta sheet and 21% beta turn structures (Osterlund, E., Eronen, I., Osterlund, K. and Vuento, M. (1985) Biochemistry 24, 2661-2667). In this work changes in the secondary structure of the protein molecule are followed as a function of different temperatures and pH values by using circular dichroic spectroscopy in far- and near-ultraviolet regions. Conformational changes are reversible when raising the temperature quickly to 55 degrees C, and then cooling slowly to 20 degrees C. A few percent of alpha-helix is apparent, when the temperature is raised to 58.5 degrees C, but only about 9% random coil is formed, when the temperature is raised up to 70 degrees C. The largest conformational change is taking place, when fibronectin samples are heated from 57 to 58.5 degrees C. According to this study more than 90% of the secondary structure of the fibronectin molecule is preserved throughout the whole temperature range studied from 20 to 70 degrees C, and this is a fact even at pH as low as 3.0, when samples are fresh and not denatured by preparative procedures.     

The purple to blue transition of bacteriorhodopsin is accompanied by a loss of the hexagonal lattice and a conformational change

X-ray diffraction measurements show that in contrast to the purple membrane, the bacteriorhodopsin molecules are not organized in a hexagonal lattice in the deionized blue membrane. Addition of Ca2+ restores both the purple color and the normal (63 A) hexagonal protein lattice. In the blue state, the circular dichroism spectrum in the visible has the typical exciton features indicating that a trimeric structure is retained. Time-resolved linear dichroism measurements show that the blue patch rotates in aqueous suspension with a mean correlation time of 11 ms and provide no evidence for rotational mobility of bacteriorhodopsin within the membrane. The circular dichroism spectra of the blue and the Ca2+-regenerated purple state in the far-UV are different, indicating a small change in secondary structure. The thermal stability of the blue membrane is much smaller than that of the purple membrane. At pH 5.0, the irreversible denaturation transition of the blue form has a midpoint at 61 degrees C. The photocycle of the blue membrane (lambda ex 590 nm) has an L intermediate around 540 nm whose decay is slowed down into the millisecond time range (5 ms). Light-dark adaptation in the blue membrane is rapid with an exponential decay time of 38 s at 25 degrees C. The purple to blue transition apparently involves a conformational change in the protein leading to a change in the aggregation state from a highly ordered and stable hexagonal lattice to a disordered array of thermally more labile trimers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protonated structures of naturally occurring deoxyribonucleic acids and their interaction with berberine

Protonation-induced conformational changes in natural DNAs of diverse base composition under the influence of low pH, low temperature, and low ionic strength have been studied using various spectroscopic techniques. At pH3.40, 10mM [Na+], and at 5 degrees C, all natural DNAs irrespective of base composition adopted an unusual and stable conformation remarkably different from the canonical B-form conformation. This protonated conformation has been characterized to have unique absorption and circular dichroic spectral characteristics and exhibited cooperative thermal melting profiles with decreased thermal melting temperatures compared to their respective B-form counterparts. The nature of this protonated structure was further investigated by monitoring the interaction of the plant alkaloid, berberine that was previously shown from our laboratory to differentially bind to B-form and H(L)-form of poly[d(G-C)] [Bioorg. Med. Chem.2003, 11, 4861]. Binding of berberine to protonated conformation of natural DNAs resulted in intrinsic circular dichroic changes as well as generation of induced circular dichroic bands for the bound berberine molecule with opposite signs and magnitude compared with B-form structures. Nevertheless, the binding of the alkaloid to both the B and protonated forms was non-linear and non-cooperative as revealed from Scatchard plots derived from spectrophotometric titration data. Steady state fluorescence studies on the other hand showed remarkable increase of the rather weak intrinsic fluorescence of berberine on binding to the protonated structure compared to the B-form structure. Taken together, these results suggest that berberine can detect the formation of significant population of H(L)-form structures under the influence of protonation irrespective of heterogeneous base compositions in natural DNAs.     

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     

Protonated forms of poly[d(G-C)] and poly(dG).poly(dC) and their interaction with berberine

The pH -induced structural changes on the conformation of homo- and hetero-polymers of guanosine-citydine (G.C) sequences were investigated using spectrophotometric and circular dichroic techniques. At pH 3.40, 10 mM [Na(+)] and 10 degrees C both polynucleotides adopted a unique and stable structural conformation different from their respective B-form structures. The protonated hetero-polymer is established as left-handed structure with Hoogsteen base pairing (H(L)-form) while the homo-polymer favored Watson-Crick base pairing with different stacking arrangements from that of B-form structure as evident from thermal melting and circular dichroic studies. The interaction of berberine, a naturally occurring protoberberine group of plant alkaloid, with the protonated structures was studied using various biophysical techniques. Binding of berberine to the H(L)-form structure resulted in intrinsic circular dichroic changes and generation of extrinsic circular dichroic bands with opposite sign and magnitude compared to its B-form structure while with the homo-polymer of G.C no such reversal of extrinsic circular dichroic bands was seen indicating different stacking arrangement of berberine at the interaction site. Scatchard analysis of the binding data, however, indicated non-cooperative binding to both the protonated forms similar to that of their respective B-form structure. Fluorescence spectral studies, on the other hand, showed remarkable increase in the intrinsic fluorescence of the alkaloid in presence of the protonated forms compared to their respective B-form structure. These results suggest that berberine could be used as a probe to detect the alteration of structural handedness due to protonation and may potentiate its use in regulatory roles for biological functions.     

Large Scale Global Structural Changes of the Purple Membrane during the Photocycle

Both the solution and the oriented film absorption and circular dichroic spectra of the bacteriorhodopsin (bR₅₆₈) and M₄₁₂ intermediate of the purple membrane photocycle were compared over the wavelength region 800-183 nm to assess structural changes during this photocycle. The main findings are (a) loss of the excitonic interaction among the chromophoric retinal transitions indicating disordering of the retinal orientations in the membrane and distortions of the membrane hexagonal crystal lattice, (b) structural change of the chromophoric retinal, (c) changes in the key interactions between the retinal and specific groups in the local environment of the apoprotein, (d) significant changes of the tertiary structure of the bR with negligible secondary structure involvement, and (e) a net tilting of the rodlike segments of the bR polypeptides away from the membrane normal. These findings are in accord with large scale global structural changes of the membrane during the photocycle and with structural metastability of the bR molecules. An important implication of these changes is the possibility of transmembrane retinal-regulated pulsating channels during the photocycle. The significance of this possibility in respect to models for the proton translocation function of this membrane is discussed.     

Circular dichroism study of the solution conformation of luteinizing hormone releasing hormone.

A systematic investigation has been made into the circular dichroic behavior of luteinizing hormone releasing hormone and its peptide fragments and deletion analogues. The results are interpreted to mean that the hormone exists in solution as an ensemble of conformers with different sensitivities to temperature and solvent composition. The far-ultraviolet circular dichroic spectra exhibited by the hormone under different experimental conditions can be simulated satisfactorily by the weighted addition of the spectra of its aliphatic- and aromatic-containing halves. However, the structure of the hormone is not simply the sum of its halves, since some conformational feature of the intact molecule perturbs the near-ultraviolet circular dichroism of its aromatic residues.     

Effects of the crystalline structure of purple membrane on the kinetics and energetics of the bacteriorhodopsin photocycle.

Time-resolved difference spectra were measured for Triton X-100 solubilized bacteriorhodopsin monomers between 100 ns and 100 ms after photoexcitation. The results are consistent with the general scheme K in equilibrium L in equilibrium M1 in equilibrium M2 in equilibrium N in equilibrium O----BR proposed previously for purple membranes [Váró, G., & Lanyi, J.K. (1990) Biochemistry 29, 2241-2250]. The rate constants which involve proton release or uptake, i.e., kLM1, kNO, and kON, were significantly higher in the monomeric protein than in purple membrane; the other steps were less affected. Analysis of the temperature dependencies of the rate constants between 5 and 30 degrees C yielded the enthalpies and entropies of activation for all steps except the two absent back-reactions. Comparison of these with data for purple membranes [Váró, G., & Lanyi, J.K. (1991) Biochemistry 30, 5016-5022] shows that the crystalline structure affects the energetics of the photocycle. In bacteriorhodopsin immobilized by the lattice of the purple membrane, the entropy changes leading to all transition states are more positive. Thus, the forward reactions proceed with less conformational hindrance. However, the thermal (enthalpic) barriers are higher. These effects are particularly pronounced for the M1----M2 and O----BR reactions. Large changes of the enthalpy and entropy levels of intermediates in the M2----BR reaction segment, but not in the K----M1 segment, upon solubilization of the protein are consistent with our earlier proposal that major protein conformational changes occur in the photocycle and they begin with the M1----M2 reaction.     

The circular dichroism of synaptosomal membranes: studies of interactions with neuropharmacological agents

The circular dichroic spectrum of synaptosomal membranes was highly reproducible and qualitatively similar to that of erythrocyte membranes. The spectrum exhibited a minimum at 224 nm, a shoulder at 212 nm and a maximum at 195 nm. The mean molar ellipticity at the maximum and minimum was approximately +8000 and –8000 respectively. The protein components were the dominant source of the CD signal. Quantitative estimates showed negligible contributions to the spectrum from cholesterol, phosphatidyl serine and TV-acyl sugars. Phospholine iodide, eserine, decamethonium, tetramethyl ammonium chloride and acetylcholine at concentrations of 10^-3 and 10^-4M did not produce detectable perturbations of the membrane circular dichroism. The circular dichroic spectrum of d-tubocurarine exhibited a maximum at 198 nm and a minimum at 212 nm. Addition of d-tubocurarine to membrane suspensions in the cuvette yielded a complex spectrum representing the result of a simple additive combination of the circular dichroic spectra of d-tubocurarine and the membranes. However, preincubation of the membranes with 3 x 10^-3m d-tubocurarine at 0-4°C for 5-10 min followed by sedimentation, several washes and resuspension resulted in a circular dichroic spectrum which appeared to involve no change in the membrane contribution, but there was a substantial decrease in the molar ellipticity of the d-tubocurarine remaining with the membranes.     

Interpretations of the effects of pH on the spectra of purple membrane.

The absorption and circular dichroism of the purple membrane in solution and the linear and circular dichroism of the purple membrane oriented in a film were used to detect changes in the membrane protein structure and membrane organization in the pH range of 2.4 to 12.6. Main findings are (a) the membrane protein structure is stable at every level of organization to pH changes over the range of 5.0 to 8.5. (b) Tertiary structural changes occur in the membrane protein structure in the pH range of 2.4 to 5.0 and 8.5 to 11.8 without any secondary structural involvement. (c) An irreversible change occurs in the membrane organization in the pH range of 11.8 to 12.6 involving large tertiary and secondary structural changes in the membrane protein. (d) The retinyl chromophore is influenced by a nearby ionizable group. (e) The membrane crystalline structure is highly stable to pH perturbation except at the high pH range of 11.0 to 11.8.     

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)     

Circular dichroism of aggregated deoxyhemoglobin S and the effect of amino acids.

It is well known that deoxyhemoglobin S (deoxy Hb S) aggregates at 37 °C and that it disaggregates at 1–5 °C. In this study solutions of pure Hb S at concentrations of 20–22 g/100 ml exhibit a normal circular dichroic spectrum in the range 250–650 nm at the temperature 1 °C. However, by the proper manipulation of the following parameters: temperatures of 1, 24 and 37 °C as well as the times required to change temperature and periods of maintaining at a certain temperature, five stages with different circular dichroic spectra can be produced. Not only the dichroic spectra of these stages are different but the kinetic behavior and stability of each of these stages are different. The evidence suggests that the mechanism of aggregation is similar to crystallization; that is, it exhibits a period of nucleation followed by growth. The overall kinetics of circular dichroic changes are described. At representative solution conditions the circular dichroic changes have been compared and found to parallel gel formation with pure Hb S. Also, the effect of certain anti-sickling amino acids (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118) on the minimum Hb S concentration at which circular dichroic changes occur has been studied, and arginine chloride and arginine aspartate were found to raise this minimum concentration appreciably.     

Interaction of lipoprotein lipase with phospholipid vesicles: effect on protein and lipid structure

The interaction of lipoprotein lipase (LpL) and a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), has been studied by several physical methods. Analysis of the circular dichroic spectrum of LpL gave the following fractional conformation: 35% alpha-helix, 30% beta-pleated sheet, and 45% remaining structure. No significant change in the circular dichroic spectrum of LpL was observed on addition of C14-ether-PC vesicles. The quenching of LpL fluorescence by acrylamide and iodide ion was decreased only slightly by addition of C14-ether-PC vesicles. Addition of LpL to sonicated C14-ether-PC vesicles containing entrapped carboxyfluorescein caused the release of less than 15% of the vesicle contents in 20 min, indicating that the enzyme did not disrupt the structure of the lipid. In contrast, greater than 80% of the vesicle contents were released with the addition of apolipoprotein A-I to an identical vesicle preparation. The temperature dependence of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into C14-ether-PC vesicles was not significantly altered by the addition of LpL. When LpL is added to vesicles, the bilayer structure of the vesicles is not disrupted as observed by freeze-fracture electron microscopy. However, at low ionic strength (0.1-0.25 M NaCl) significant aggregation of intact vesicles is observed by light scattering and electron microscopy. Vesicle aggregation is prevented and reversed by 1 M NaCl and by heparin. These data demonstrate that LpL binds to the surface of a lipid interface, without dramatic changes in lipid bilayer or protein structure.     

The contribution of electrostatic factors to the stabilization of the conformation of cytochrome c. Studies on the maleylated protein.

All the lysines of horse heart cytochrome c were maleylated yielding a low spin product. At room temperature and low salt concentration, this product lacked the 695 nm absorption band and showed tryptophan fluorescence and circular dichroic spectra typical of denatured cytochrome c. The 695 nm band and the native tryptophan fluorescence and circular dichroic spectra were restored by addition of salts, their effectiveness being dependent on the charge of the cation. On low salt concentration, the 695 nm band was also restored by lowering the temperature. Studies of the temperature dependence of the 695 nm band indicate that the thermal denaturation of maleylated cytochrome c occurs at temperatures 60-70 degrees C lower than in the native protein. This implies a destabilization of the native conformation by 5.6 kcal/mol; a similar value is evidenced by comparative urea denaturation studies on the native and modified proteins. The results confirm the assumption that the native conformation of cytochrome c is mostly determined by interactions involving internal residues.