The surface potential on the purple membrane measured using a modified bacteriorhodopsin chromophore as the spectroscopic probe

The surface potential of the purple membrane was measured by a novel method by using an artificial bacteriorhodopsin whose chromophore was 13-CF₃ retinal instead of retinal. When attached to the apoprotein by a Schiff base, the intrinsic pK of the 13-CF₃ chromophore is around 7.3. The apparent p_K of this pigment depends on the surface potential and thus on the electrolyte concentration. This allowed us to determine the surface charge density using the Gouy-Chapman equation. The surface charge density was found to be −1.65 ± 0.15 × 10⁻³ electronic charges per Ų or about 2 negative charges/bacteriorhodopsin. This large value for the surface potential probably explains both part of the strong apparent association of divalent cations with the membrane and the effect of low salt concentrations on light-induced proton release from the purple membrane.     

Studies on the surface structure of the intracytoplasmic membrane in the photosynthetic purple bacterium Chromatium vinosum by means of chemical modification

The chemical and physical properties of bullfrog serum low density lipoprotein (LDL) were investigated. On a weight percentage basis, LDL contained cholesterol ester, 30.3%; cholesterol, 5.6%; triglyceride, 12.5%; phospholipids, 23.3%; and protein, 22.4%. The fatty acid compositions of triglycerides and major phospholipids from the bullfrog serum LDL were quite similar to those of human serum LDL. However, the fatty acid composition of the chlesterol ester from the bullfrog serum LDL was quite different from that of the human serum LDL. The average particle weight, determined by gel filtration, was 2 X 10(6) daltons. This value is very close to that of human LDL. In the fluorescence emission spectrum of bullfrog serum LDL, the emission maximum was 324 nm. The amino acid composition of the apo-LDL resembled that of human apo-LDL.     

Influence of the lipid membrane environment on structure and activity of the outer membrane protein Ail from Yersinia pestis

The surrounding environment has significant consequences for the structural and functional properties of membrane proteins. While native structure and function can be reconstituted in lipid bilayer membranes, the detergents used for protein solubilization are not always compatible with biological activity and, hence, not always appropriate for direct detection of ligand binding by NMR spectroscopy. Here we describe how the sample environment affects the activity of the outer membrane protein Ail (attachment invasion locus) from Yersinia pestis. Although Ail adopts the correct β-barrel fold in micelles, the high detergent concentrations required for NMR structural studies are not compatible with the ligand binding functionality of the protein. We also describe preparations of Ail embedded in phospholipid bilayer nanodiscs, optimized for NMR studies and ligand binding activity assays. Ail in nanodiscs is capable of binding its human ligand fibronectin and also yields high quality NMR spectra that reflect the proper fold. Binding activity assays, developed to be performed directly with the NMR samples, show that ligand binding involves the extracellular loops of Ail. The data show that even when detergent micelles support the protein fold, detergents can interfere with activity in subtle ways.     

Effects of membrane potential and surface potential on the kinetics of solute transport

A theoretical study has been made of the influence of the transmembrane potential difference and the surface potential of living cells on the kinetics of carried-mediated solute transport. It is assumed that the form of the free energy barrier within the membrane may be approximated by one dominant symmetrical peak, and that the electrical field is constant. Both single-ion transport kinetics and cotransport of an ion with a neutral solute are dealt with. Provided that the surface potential and the transmembrane potential are constant, the concentration dependence of the uptake rate is given by the Michaelis-Menten equation. The kinetic parameters, the maximal rate of uptake and the K_m, depend on both the surface potential and the membrane potential in a rather complex way. It is shown that the intuitive notion, that the maximal rate of cation uptake will increase when the cell membrane is hyperpolarized, is wrong in its generallity. Both an increase or a decrease may occur, depending on the characteristics of the transport system involved. If the magnitude of the membrane potential and the surface potential depends on the substrate concentration, marked deviations from Michaelis-Menten kinetics may come to the fore. This may result in either apparent positive or apparent negative homotrope cooperative effects. Enhancement of the uptake rate of the substrate ion may occur on adding another cation, despite the fact that the membrane will become depolarized. The same type of complex transport kinetics as found for Rb⁺ and Na⁺ uptake in yeast cells can be simulated by using a single-site transport model and including effects of the membrane potential.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium.

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

Influence of structure,pH and membrane potential on proton movement in cytochrome oxidase

Cytochrome c oxidase (CcO) reconstituted into phospholipid vesicles and subject to a membrane potential, exhibits different characteristics than the free enzyme, with respect to effects of mutations, pH, inhibitors, and native structural differences between CcO from different species. The results indicate that the membrane potential influences the conformation of CcO and the direction of proton movement in the exit path. The importance of the protein structure above the hemes in proton exit, back leak and respiratory control is discussed.     

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.     

Influence of membrane lipids on the photochemistry of bacteriorhodopsin in the purple membrane of Halobacterium halobium

Purple membrane fragments from Halobacterium halobium were reconstituted with the native lipids replaced by dipalmitoyl phosphatidylcholine and by egg lecithin. In parallel studies the temperature dependence of bacteriorhodopsin phototransient lifetime and absorption dichroism and of in situ lipid microviscosity were determined; the former two by, respectively, conventional and polarization flash photometry, and the latter by observation of emission depolarization of an embedded fluorescent dye, 1,6-diphenyl-1,3,5-hexatriene. Discontinuities in lipid microviscosity profiles in native and egg lecithin purple membrane were reflected in both the photochemical cycle frequency and bacteriorhodopsin chromophore rotational mobility. The influence exerted by membrane-lipid viscosity appears to be a secondary effect, and points to the bacteriorhodopsin chromophoric group being situated in the protein interior.     

Influence of the surface potential on the Michaelis constant of membrane-bound enzymes: effect of membrane solubilization

We have purified from a membrane fraction of bovine brain a calmodulin-binding protein (calspectin) that shares a number of properties with erythrocyte spectrin: It has a heterodimeric structure with M_r 240 000 and 235 000 and binds to (dimeric form) or crosslinks (tetrameric form) F-actin. We show that calspectin (tetramer) is capable of inducing the polymerization of G-actin to actin filaments by increasing nucleation under conditions where actin alone polymerizes at a much slower rate. Thus, brain calspectin behaves in the same manner as erythrocyte spectrin, supporting the idea that, in conjunction with actin oligomers it comprises the cytoskeletal meshwork underlying the cytoplasmic surface of the nerve cell.     

Bacteriorhodopsin in ice. Accelerated proton transfer from the purple membrane surface

The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H(+)-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.     

Donnan potential and surface potential of a charged membrane.

A model is presented for the electrical potential distribution across a charged biological membrane that is in equilibrium with an electrolyte solution. We assume that a membrane has charged surface layers of thickness d on both surfaces of the membrane, where the fixed charges are distributed at a uniform density N within the layers, and that these charged layers are permeable to electrolyte ions. This model unites two different concepts, that is, the Donnan potential and the surface potential (or the Gouy-Chapman double-layer potential). Namely, the present model leads to the Donnan potential when d much greater than 1/k' (k' is the Debye-Hückel parameter of the surface charge layer) and to the surface potential as d----0, keeping the product Nd constant. The potential distribution depends significantly on the thickness d of the surface charge layer when d less than or approximately equal to 1/k'.     

Effects of surface potential and membrane potential on the midpoint potential of cytochrome c-555 bound to the chromatophore membrane of Chromatium vinosum

The values of midpoint potential (Em) of cytochrome c-555 bound to the chromatophore membranes of a photosynthetic bacterium Chromatium vinosum was determined under various pH and salt conditions. After a long incubation at high ionic concentrations in the presence of carbonylcyanide m-chlorophenylhydrazone, which was added to abolish electrical potential difference between the inner and outer bulk phases of chromatophore, the Em value was almost constant at pH values between 4.0 and 8.4. With the decrease of salt concentration, the pH dependence of the Em value became more marked. Under low ionic conditions, Em became more positive with the decrease of pH. Addition of salt made the value more positive or negative at pH values higher or lower than 4.5, respectively. Divalent cation salts were more effective than monovalent cation salts in producing the positive shift of Em at pH 7.8. The Em value became more positive when the electrical potential of the inner side of the chromatophore was made more positive by the diffusion potential induced by the K+ concentration gradient in the presence of valinomycin. These results were explained by a change of redox potential at the inner surface of the chromatophore membrane, at which the cytochrome is assumed to be situated, due to the electrical potential difference with respect to the outer solution induced by the surface potential or membrane potential change. The values for the surface potential and the net surface charge density of the inner surface of the chromatophore membrane were estimated using the Gouy-Chapman diffuse double layer theory.     

Pressure effects on the photocycle of purple membrane

We studied the effects of hydrostatic pressure on the kinetics of the photocycle of purple membrane from Halobacterium halobium. The data were interpreted in terms of a unidirectional and unbranched model. We found that all of the distinct processes of the photocycle are retarded by pressure, with the earlier, fast processes showing less sensitivity to pressure than the later, slow processes. The qualitative similarity of these results with the effects of solvent viscosity on the photocycle kinetics suggests that the primary effects of pressure on the kinetics are via the intrinsic viscosity of the membrane and not via activation volumes. There is a strong quantitative correlation between the pressure effects and the solvent viscosity effects, further supporting this interpretation. We observed a monotonic decrease in the positive absorbance change signal at 640 nm near the end of the photocycle as the pressure is increased. This signal is usually ascribed to the O intermediate, and we interpreted our finding, along with evidence from other experiments, to mean that an ionizable group or groups, such as carboxylic acids, are undissociated and uncharged in O.     

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.     

Effects of surfactant-associated protein SP-B synthetic analogs on the structure and surface activity of model membrane bilayers.

The effect of several synthetic peptides based on the sequence of human pulmonary surfactant-associated protein B (SPB) on the molecular packing of model membrane lipids (7:1 dipalmitoyl phosphatidylcholine (DPPC)/dipalmitoyl phosphatidylglycerol (DPPG)) was studied using fluorescence anisotropy. This information was then correlated with complementary biophysical data obtained on both a modified Wilhelmy-Langmuir balance and a pulsating bubble surfactometer. The SP-B peptides examined in these studies are synthetic human SP-B Phe1-Ser78 (SP-B 1-78, full-length sequence), synthetic human SP-B Phe1-Thr60 (SP-B 1-60), synthetic human SP-B Phe1-Ala20 (SP-B 1-20), synthetic human SP-B Ala20-Thr60 (SP-B 20-60), synthetic human SP-B Leu27-Ser78 (SP-B 27-78), synthetic human SP-B Leu40-Thr60 (SP-B 40-60) and synthetic human SP-B Tyr53-Ser78 (SP-B 53-78). trans-parinaric acid was utilized to detect changes in ordering of lipids within the interior upon incorporation of synthetic SP-B peptide, whereas 1-hexadecanoyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)-a min ohexanoyl] phosphatidylcholine (6-NBD-PC) and 1-acyl-2-[N-(7-nitro-2-benzoxa-1,3-diazol-4-yl)aminohexanoyl ] phosphatidylglycerol (6-NBD-PG) were utilized to determine alterations in lipid order at the surface of the model membrane bilayer. With the exception of SP-B 40-60, which corresponds to the most hydrophobic segment of the full-length SP-B, none of the other peptide significantly perturbed the interior bilayer as determined by fluorescence anisotropy of trans-parinaric acid. Incorporation of any of the peptides with the exception of SP-B 40-60, resulted in an increase in anisotropy of NBD-PC. The most significant enhancements resulted from the addition of SP-B 1-78, SP-B 1-20, SP-B 27-78 or SP-B 53-78. The magnitude of anisotropy increase with these peptides is similar to that observed with an equivalent molar ratio of native SP-B isolated from a bovine source. These observations suggest that these four synthetic peptides have the structural and compositional characteristics required for surface ordering of the membrane bilayer in a manner similar to that observed with native SP-B, thereby facilitating the surfactant-like properties of phospholipid mixtures.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Inhibition of lipoprotein lipase activity by sphingomyelin: role of membrane surface structure

We have recently shown that sphingomyelin (SM) strongly inhibits lipoprotein lipase (LPL)-mediated lipolysis in monolayers and emulsion particles. To further evaluate how SM modulates LPL activity on the emulsion surface, the relationship between membrane surface structure and LPL activity was investigated. We measured fluorescence anisotropy of 1-palmitoyl-2-[3-(diphenylhexatrienyl)propionyl]-sn-3-phosphati dylcho line, probing surface acyl chain fluidity, and fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine in H(2)O and D(2)O buffer, assessing the degree of hydration in the head group region. The results revealed that incorporation of egg SM into triolein-egg phosphatidylcholine emulsions markedly increased acyl chain order and decreased head group hydration of the surface monolayers. In contrast, cholesterol was shown to increase head group hydration despite a strong increase in acyl chain order. The close correlation between the apparent K(m) values of LPL and the degree of head group hydration indicated that LPL interacts with the head group region rather than with the hydrophobic interior of the surface monolayers. However, apparent V(max) did not show a simple correlation with any surface structure, and the finding in which SM had no effect on apparent V(max) of medium-chain triglyceride emulsions suggested that the hydrophobic interaction between acyl chains of SM and triglyceride at the emulsion surface is important for determining the apparent V(max). These results showed conclusively that SM inhibits LPL activity mainly by changing the emulsion surface structure and not by a specific interaction between SM and LPL.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.