Solubilization and structural stability of bacteriorhodopsin with a mild nonionic detergent, n-Octyl-β-thioglucoside

Solubilization and structural stability of a membrane protein bacteriorhodopsin (bR) with n-octyl-β-thioglucoside (OTG) was investigated in comparison with a previous study on bR solubilized with n-octyl-β-glucoside (OG). Highly efficient and stable solubilization of bR with OTG was accomplished above the OTG concentration of about 15 mM. In comparison with OG-solubilized bR, the structural stability of OTG-solubilized bR was high in the dark and under light illumination. These results indicate that OTG is a detergent superior to OG for solubilizing bR molecules.     

Solubilization and Structural Stability of Bacteriorhodopsin with a Mild Nonionic Detergent,n-Octyl-β-thioglucoside

Solubilization and structural stability of a membrane protein bacteriorhodopsin (bR) with n-octyl-β-thioglucoside (OTG) was investigated in comparison with a previous study on bR solubilized with n-octyl-β-glucoside (OG). Highly efficient and stable solubilization of bR with OTG was accomplished above the OTG concentration of about 15 mM. In comparison with OG-solubilized bR, the structural stability of OTG-solubilized bR was high in the dark and under light illumination. These results indicate that OTG is a detergent superior to OG for solubilizing bR molecules.     

Formation of unilamellar lipid vesicles of controllable dimensions by detergent dialysis.

Vesicles are formed by solubilizing mixtures of phosphatidylcholine and cholesterol with sodium cholate and removing the detergent by rapid (hollow fiber) dialysis [e.g., Goldin, S. M. (1977) J. Biol. Chem. 252, 5630--5642]. Characterization of the vesicle size distribution by agarose gel filtration, and determination of the intravesicular aqueous compartment, demonstrates that the vesicles are relatively homogeneous in size and are primarily unilamellar. The mean diameter of the vesicles can be varied from 340 to 1280 A by varying the conditions under which they are formed; increasing the mole fraction of cholesterol and lowering the pH of the dialysate tend to produce larger vesicles. The gentle detergent treatment required for vesicle formation and the ability to control vesicle size distribution reproducibly may make this method particularly useful in studies of reconstitution of membrane proteins and in use of vesicles as vehicles for delivery of materials to living cells.     

Large scale nonproton ion release and bacteriorhodopsin's state of aggregation in lipid vesicles. I. Monomers.

Light-induced conductivity transients have been observed in preparations of bacteriorhodopsin (bR) in phospholipid vesicles at high lipid/protein molar ratios. Under these conditions, bR is known to be dissolved as monomers in the lipid bilayer. The conductivity transients are due mostly to proton movements, including a trans-membrane component. Kinetic resolution of the conductance change due to proton ionophore-induced leakage through the vesicle membrane provides a novel method to quantitate the number of protons pumped, even in heavily buffered solutions. Some of the transient signal seen on the timescale of the bR photocycle is due to nonproton ions but is smaller than that observed in native purple membranes at pH 7 in low salt. Furthermore, when the pH is raised to 8, the very large transient nonproton ion release seen in purple membranes is not seen in the vesicles. This correlates well with previous results (Marinetti, T., and D. Mauzerall, 1986, Biophys. J., 50:405-415), in which the nonproton ion movements observed with native purple membranes were abolished by solubilization in Triton X-100. Thus, the nonproton ion release appears to be a property of bR in the native aggregated state.     

Effect of a light-induced pH gradient on purple-to-blue and purple-to-red transitions of bacteriorhodopsin

Bacteriorhodopsin-containing vesicles that were able to alkalize the extravesicular medium by greater than 1.5 pH units under illumination, i.e., inside-out vesicles, were reconstituted by reverse-phase evaporation with Halobacterium halobium polar lipids or exogenous phospholipids. Acid titration of a dark-adapted sample was accompanied by a color change from purple to blue (pKa = 2.5-4.5 in 0.15 M K2SO4), and alkali titration resulted in the formation of a red species absorbing maximally at 480 nm (pKa = 7 to greater than 9), the pKa values and the extents of these color changes being dependent on the nature of lipid. When a vesicle suspension at neutral or weakly acidic pH was irradiated by continuous light so that a large pH gradient was generated across the membrane, either a purple-to-blue or a purple-to-red transition took place. The light-induced purple-to-red transition was significant in an unbuffered vesicle suspension and correlated with the pH change in the extravesicular medium. The result suggests that the purple-to-red transition is driven from the extravesicular side, i.e., from the C-terminal membrane surface. In the presence of buffer molecules outside, the dominant color change induced in the light was the purple-to-blue transition, which seemed to be due to a large decrease in the intravesicular pH. But an apparently inconsistent result was obtained when the extravesicular medium was acidified by a HCl pulse, which was accompanied by a rapid color change to blue. We arrived at the following explanation: The two bR isomers, one containing all-trans-retinal and the other 13-cis-retinal, respond differently to pH changes in the extravesicular and the intravesicular medium. In this relation, full light adaptation was not achieved when the light-induced purple-to-blue transition was significant; i.e., only the 13-cis isomer is likely to respond to a pH change at the N-terminal membrane surface.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side.

In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21 °C.

In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds.

Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds.

The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.     

Triton X－100对紫膜蛋白的效应

本文采用同步辐射小角Ｘ射线散射方法研究了用非离子表面活性剂ＴｒｉｔｏｎＸ—１００处理后的嗜盐菌紫膜及其视紫红质蛋白结构的变化。实验结果表明,用不同浓度的ＴｒｉｔｏｎＸ—１００处理紫膜碎片时,紫膜及其蛋白所处的状态有着很大变化。     

Membrane curvature affects the stability and folding kinetics of bacteriorhodopsin

Biological membrane is crucial for the function, stability and folding of membrane proteins. By studying the stability and folding kinetics of bacteriorhodopsin (bR) in lipid vesicles with different sizes, here we report the influence of membrane curvature (vesicle size) on the stability and folding kinetics of bR. The results show that both the stability and folding kinetics of bR can be significantly changed when reconstituted into mimic membranes with different curvatures. The stability of bR decreases and unfolding rate of bR increases with the growth of vesicle size, i.e. decrease of membrane curvature. Our results suggest that it is possible to regulate the properties of membrane proteins by changing the curvature of membranes.     

Time-resolved absorbance changes induced by fast acidification of bacteriorhodopsin in vesicle systems.

The direction of the accessibility to protons of the binding site in bacteriorhodopsin is of primary importance in elucidating the proton-pump mechanism. The problem is approached via the pH-dependent equilibrium bR560 in equilibrium bR605 in vesicles with preferentially oriented purple membranes. Fast acidification (stopped-flow) experiments with inside-out, monomeric, bR vesicles were carried out with and without a buffer enclosed in the vesicle interior. The results, showing a buffer-induced delay in the formation of bR605, indicate that the binding site is accessible to protons from the inside of the vesicles. We arrive at this conclusion also by working with inside-out trimeric vesicles in the presence and in the absence of H+ (and K+) ionophores. The results suggest that in Halobacterium halobium, the binding site and thus the retinal Schiff base are exposed to the outside of the cell. This conclusion is consistent with a pumping mechanism based on a light-induced pK change.     

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

Phospholipid vesicle formation using nonionic detergents with low monomer solubility. Kinetic factors determine vesicle size and permeability

The method developed previously for formation of unilamellar vesicles from mixed micelles of egg lecithin and octyl glucoside [Mimms, L. T., Zampighi, G., Nozaki, Y., Tanford, C., & Reynolds, J. A. (1981) Biochemistry 20, 833-840] has been extended to allow for (1) use of nonionic detergents with much lower critical micelle concentrations and (2) variation in the time course of detergent removal. The results demonstrate the importance of kinetic factors, especially in the determination of vesicle size: initially formed vesicles are small, but the size increases slowly thereafter if detergent is not removed too quickly. Vesicle size remains fixed when the molar detergent/lipid ratio falls below about 1/1, and detergent removal becomes increasingly difficult thereafter, presumably because flip-flop of detergent from the inner to the outer leaflet of the bilayer membrane is very slow. Residual detergent (to about 25 mol %) has surprisingly little effect on anion permeability but increases cation permeability to the point where the normal discrimination between anions and cations (in pure lipid vesicles) is lost. Detergent added to initially detergent-free vesicles readily partitions into vesicular membranes (presumably only into the outer leaflet) and has a qualitatively similar effect on permeability. Vesicles produced by this method, regardless of residual detergent level, were found to be predominantly unilamellar: no multilamellar liposomes or other lipid aggregates could be detected within the accuracy of the methods employed.     

Effective encapsulation of proteins into size-controlled phospholipid vesicles using freeze-thawing and extrusion

We are aiming to improve the encapsulation efficiency of proteins in a size-regulated phospholipid vesicle using an extrusion method. Mixed lipids (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-dipalmitoyl-l-glutamate-N-succinic acid (DPEA), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5,000)] (PEG-DSPE) at a molar ratio of 5, 5, 1, and 0.033 were hydrated with a NaOH solution (7.6 mM) to obtain a polydispersed multilamellar vesicle dispersion (50 nm to 30 microm diameter). The polydispersed vesicles were converted to smaller vesicles having an average diameter of ca. 500 nm with a relatively narrow size distribution by freeze-thawing at a lipid concentration of 2 g dL(-)(1) and cooling rate of -140 degrees C min(-1). The lyophilized powder of the freeze-thawed vesicles was rehydrated into a concentrated protein solution (carbonyl hemoglobin solution, 40 g dL(-1)) and retained the size and size distribution of the original vesicles. The resulting vesicle dispersion smoothly permeated through the membrane filters during extrusion. The average permeation rate of the freeze-thawed vesicles was ca. 30 times faster than that of simple hydrated vesicles. During the extrusion process, proteins were encapsulated into the reconstructed vesicles with a diameter of 250 +/- 20 nm.     

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.     

Determination of bilayer membrane bending stiffness by tether formation from giant, thin-walled vesicles.

The curvature elastic modulus (bending stiffness) of stearoyloleoyl phosphatidylcholine (SOPC) bilayer membrane is determined from membrane tether formation experiments. R. E. Waugh and R. M. Hochmuth 1987. Biophys. J. 52:391-400) have shown that the radius of a bilayer cylinder (tether) is inversely related to the force supported along its axis. The coefficient that relates the axial force on the tether to the tether radius is the membrane bending stiffness. Thus, the bending stiffness can be calculated directly from measurements of the tether radius as a function of force. Giant (10-50-microns diam) thin-walled vesicles were aspirated into a micropipette and a tether was pulled out of the surface by gravitational forces on small glass beads that had adhered to the vesicle surface. Because the vesicle keeps constant surface area and volume, formation of the tether requires displacement of material from the projection of the vesicle in the pipette. Tethers can be made to grow longer or shorter or to maintain equilibrium by adjusting the aspiration pressure in the micropipette at constant tether force. The ratio of the change in the length of the tether to the change in the projection length is proportional to the ratio of the pipette radius to the tether radius. Thus, knowing the density and diameter of the glass beads and measuring the displacement of the projection as a function of tether length, independent determinations of the force on the tether and the tether radius were obtained. The bending stiffness for an SOPC bilayer obtained from these data is approximately 2.0 x 10(-12) dyn cm, for tether radii in the range of 20-100 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bacteriorhodopsin photoreaction: identification of a long-lived intermediate N (P,R350) at high pH and its M-like photoproduct

An alkaline suspension of light-adapted purple membrane exposed to continuous light showed a large absorption depletion at 580 nm and a small increase around 350 nm. We attribute this absorption change to an efficient photoconversion of bR570 into a photoproduct N (P,R350), which has a major absorption maximum between 550 and 560 nm but has lower absorbance than bR570. N was barely detectable at low pH, low ionic strength, and physiological temperature. However, when the thermal relaxation of N to bR570 was inhibited by increasing pH, increasing ionic strength, and decreasing temperature, its relaxation time could be as long as 10 s at room temperature. N is also photoactive; when it is present in significant concentrations, e.g., accumulated by background light, the flash-induced absorption changes of purple membrane suspensions were affected. Double-excitation experiments showed an M-like photoproduct of N,NM, with an absorption maximum near 410 nm and a much longer lifetime than M412. It may be in equilibrium with an L-like precursor NL. We suggest that N occurs after M412 in the photoreaction cycle and that its photoproduct NM decays into bR570. Thus, at high pH and high light intensity, the overall photoreaction of bR may be approximated by the two-photon cycle bR570----M412----N----(NL----NM)----bR570, whereas at neutral pH and low light intensity it can be described by the one-photon cycle bR570----M412----N----O640----bR570.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanical properties of vesicles. I. Coordinated analysis of osmotic swelling and lysis.

To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles. Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies. Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution. Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer. Such vesicles could not be used as model systems for the analysis of membrane mechanical properties. NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar. Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye. Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy. These experiments and the accompanying analysis (Hallett, F.R., J. Marsh, B.G. Nickel, and J.M. Wood. 1993. Biophys. J. 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.     

Electroporative deformation of salt filled lipid vesicles

Membrane electroporation, vesicle shape deformation and aggregation of small, NaCl-filled lipid vesicles (of radius a = 50 nm) in DC electric fields was characterized using conductometric and turbidimetrical data. At pulse durations t_E≤ 55 ± 5 ms the increase in the conductivity of the vesicle suspension is due to the field-induced efflux of electrolyte through membrane electropores. Membrane electroporation and Maxwell stress on the vesicle membrane lead to vesicle elongation concomitant with small volume reduction (up to 0.6% in an electric field of E = 1 MV m^–1). At t_E > 55 ± 5 ms, further increases in the conductivity and the optical density suggest electroaggregation and electrofusion of vesicles. The conductivity changes after the electric pulse termination reflect salt ion efflux through slowly resealing electropores. The analysis of the volume reduction kinetics yields the bending rigidity κ = (4.1 ± 0.3) ⋅ 10^–20 J of the vesicle membrane. If the flow of Na⁺ and Cl^– ions from the vesicle interior is treated in terms of Hagen-Poiseuille's equation, the number of permeable electropores is N = 39 per vesicle with mean pore radius r_p = 0.85 ± 0.05 nm at E = 1 MVm^–1 and t_E≤ 55 ± 5 ms. The turbidimetric and conductometric data suggest that small lipid vesicles (a ≤ 50 nm) are not associated with extensive membrane thermal undulations or superstructures. In particular with respect to membrane curvature, the vesicle results are suggestive for the design and optimization of electroporative delivery of drugs and genes to cell tissue at small field strengths (≤1 MVm^–1) and large pulse durations (≤100 ms). Received: 8 July 1997 / Accepted: 15 September 1997     

Vesicle size distributions measured by flow field-flow fractionation coupled with multiangle light scattering.

The separation method, flow field-flow fractionation (flow FFF), is coupled on-line with multiangle laser light scattering (MALLS) for simultaneous measurement of the size and concentration of vesicles eluting continuously from the fractionator. These size and concentration data, gathered as a function of elution time, may be used to construct both number- and mass-weighted vesicle size distributions. Unlike most competing, noninvasive methods, this flow FFF/MALLS technique enables measurement of vesicle size distributions without a separate refractive index detector, calibration using particle size standards, or prior assumptions about the shape of the size distribution. Experimentally measured size distributions of vesicles formed by extrusion and detergent removal are non-Gaussian and are fit well by the Weibull distribution. Flow FFF/MALLS reveals that both the extrusion and detergent dialysis vesicle formation methods can yield nearly size monodisperse populations with standard deviations of approximately 8% about the mean diameter. In contrast to the rather low resolution of dynamic light scattering in analyzing bimodal systems, flow FFF/MALLS is shown to resolve vesicle subpopulations that differ by much less than a factor of two in mean size.