Maarten P. Heyn

Determination of lipid order parameters and rotational correlation times from fluorescence depolarization experiments

In discussing the properties of membrane lipids it is important to keep concepts related to the orientational order and to the dynamics of these molecules apart. The average conformation of the acyl chain or the order parameter of a segment are examples of static or time-averaged quantities. The rotational correlation time of a chain segment, its mobility or the viscosity it experiences during motion are notions which belong in the class of dynamic concepts. In many applications of spin label and fluorescence probes the terminology has become rather imprecise and concepts from these two areas have been used interchangeably. The word fluidity for example has been used with connotations of both order and motion. Until recently steady-state fluorescence depolarization measurements with fluorescent probes like 1,6diphenyl-1,3 ,.5-hexatriene(DPH) have been interpreted exclusively in terms of a microviscosity, i.e., given a dynamic interpretation. In a number of important contributions [l-4] it was shown that the rotational motion of DPH around axes perpendicular to its long axis is restricted and that the equilibrium orientational distribution of the probe is anisotropic. As shown below these results imply that the steadystate fluorescence anisotropy contains not only information on the dynamics (viscosity) but also on the statics (order) of the label and that the simple interpretation in terms of a microviscosity has to be revised. It is the purpose of this paper to contribute to the interpretation of fluorescence depolarization experiments with fluorescent lipid probes and to clarify the concepts involved. A systematic approach is outlined

A natural CD label to probe the structure of the purple membrane from Halobacterium halobium by means of exciton coupling effects.

Coupling between retinal chromophores on adjacent bacteriorhodopsin molecules in the hexagonal surface lattice of the purple membrane from Halobacterium halobium R1 leads to exciton circular dichroism (CD) spectra in the 567 nm absorption band. Uncoupling by solubilization of bacteriorhodopsin in Triton X-100 results in a loss of this couplet. In dimethylsulfoxide (DMSO)/water mixtures, both exciton peaks disappear and a positive CD band develops at 460 nm. The change is reversible. It is suggested that DMSO increases the protein mobility in the membrane. Bleaching causes the concerted disappearance of both exciton peaks around 567 nm, and the appearance of optical activity in the 412 nm band. Because of its strong dependence on geometry, this CD effect appears to be a sensitive probe to study changes in protein mobility and in protein-protein interactions.

Transient and linear dichroism studies on bacteriorhodopsin: Determination of the orientation of the 568 nm all-trans retinal chromophore☆

Abstract The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways. In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined. In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °. No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977). The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.

Temperature-dependent aggregation of bacteriorhodopsin in dipalmitoyl- and dimyristoylphosphatidylcholine vesicles*

Bacteriorhodopsin has been incorporated into large unilamellar lipid vesicles. Its aggregation behaviour was investigated using X-ray diffraction, electron microscopy, circular dichroism and rotational diffusion measurements. At temperatures below the lipid phase transition, bacteriorhodopsin crystallizes into patches with the same hexagonal lattice observed in the purple membrane. Above the phase transition, the lattice disaggregates and the protein molecules are monomeric provided the lipid to protein ratio is sufficiently high.

Light-induced Proton Release of Phytochrome Is Coupled to the Transient Deprotonation of the Tetrapyrrole Chromophore

The Pr → Pfr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-RC were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in Pr and ZZEssa in Pfr, with all pyrrole nitrogens being protonated. Photoexcitation of Pr is followed by (at least) three thermal relaxation components in the formation of Pfr with time constants of 230 μs and 3.1 and 260 ms. H2O/D2O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-RC, respectively. Resonance Raman measurements of Meta-RC indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-RC are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the Pr → Pfr photocon-version mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the Pfr formation that switch between the active and the inactive state of the photoreceptor.

[2] Preparation and properties of monomeric bacteriorhodopsin

Publisher Summary This chapter discusses the preparation and properties of monomeric bacteriorhodopsin (BR). The BR molecules in the purple membrane (PM) are organized into a two-dimensional hexagonal lattice of trimers that are surrounded by about 30 lipid molecules. To study the structure and function of isolated BR molecules, it is advantageous to solubilize the PM into BR monomers. These BR monomers can be reconstituted with exogenous phospholipids to form vesicles and planar bilayer membranes. They can also be reassembled into two- and three-dimensional crystals. Till date, Triton X-100, and octyl-β-D-glucoside are the only detergents that fulfil the essential criteria for this purpose. They solubilize the PM to the state of monomers without significantly affecting the spectral and functional properties and can be removed again if desired, Other detergents tested—Ammonyx LO, cetyltrimethylammonium bromide, cholate, deoxycholate, digitonin, dodecyl trimethylammonium bromide, Emulphogene, and Tween 80—either denature BR or do not yield monomers.

Highly conserved residues Asp-197 and His-250 in agp1 phytochrome control the proton affinity of the chromophore and Pfr formation

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pKa from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photo-induced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc → Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

Lateral segregation of proteins induced by cholesterol in bacteriorhodopsin-phospholipid vesicles

Bacteriorhodopsin in dimyristoylphosphatidylcholine vesicles is randomly distributed in the plane of the membrane and exhibits rotational diffusion above the gel to liquid-crystalline phase transition. Incorporation of cholesterol results in loss of rotational mobility of bacteriorhodopsin, which on the basis of electron microscopy and CD measurements can be assigned to the formation of protein aggregates. It is concluded that bacteriorhodopsin is soluble in the fluid phosphatidylcholine phase but segregates when cholesterol is present in the lipid bilayer.

Distributed kinetics of the charge movements in bacteriorhodopsin: evidence for conformational substates.

The flash-induced charge movements during the photocycle of light-adapted bacteriorhodopsin in purple membranes attached to a black lipid membrane were investigated under voltage clamp and current clamp conditions. Signal registration ranged from 200 ns to 30 s after flash excitation using a logarithmic clock, allowing the equally weighted measurement of the electrical phenomena over eight decades of time. The active pumping signals were separated from the passive system discharge on the basis of an equivalent circuit analysis. Both measuring methods were shown to yield equivalent results, but the charge translocation could be accurately monitored over the whole time range only under current clamp conditions. To describe the time course of the photovoltage signals a model based on distributed kinetics was found to be more appropriate than discrete first order processes suggesting the existence of conformational substates with distributed activation energies. The time course of the active charge displacement is characterised by a continuous relaxation time spectrum with three broad peaks plus an unresolved fast transient (<0.3 mus) of opposite polarity. The time constants and relative amplitudes (in brackets) derived from the peak rate constants and relative areas of the three bands are: tau(1) = 32 mus (20%), tau(2) = 0.89 ms (15%) and tau(3) = 18 ms (65%) at 25 degrees C in 150 mM KCl at pH7. The Arrhenius plots of the peak rate constants were linear yielding activation energies of E(A1) = 57 kJ/mol, E(A2) = 52 kJ/mol, and E(A3) = 44 kJ/mol. The electrical signal at 890 mus has no counterpart in the photocycle of bacteriorhodopsin suspensions. Fits with a sum of exponentials required 5 to 6 components and were not reproducible. Analysis of photoelectrical signals with continuous relaxation time spectra gave equally good fits with fewer parameters and were well reproducible.

Evidence for the first phase of the reprotonation switch of bacteriorhodopsin from time-resolved photovoltage and flash photolysis experiments on the photoreversal of the M-intermediate

The kinetics of the photoreversal reaction of the M-intermediate of bacteriorhodopsin (bR) was investigated by time-resolved optical absorption spectroscopy and photovoltage measurements using double-flash excitation (a green flash (532 nm) followed by a blue flash (400 nm) after a variable delay). The sign of the photovoltage and the 1H/2H kinetic isotope effect indicate that the Schiff base is reprotonated by a group between the Schiff base and the extracellular surface, probably Asp85. Analysis of the kinetic data shows that the charge movement in 150 mM KCl at 12 degrees C is characterized by two components with time constants of approximately 100 ns and approximately 600 ns, respectively, which are independent of the delay time between the flashes and the pH. The amplitudes of the fast and slow components depend on the delay and the pH. The slower component starts to contribute to the charge movement only after delays longer than 100 micros, is absent at low pH, and increases in amplitude with a pKa of approximately 6. Because the proton release group deprotonates after 70-100 micros and has a transient pKa of 5.8, these results suggest the following assignment: the fast and the combination of fast and slow components represent photoreversal from two M states, with the release group protonated and deprotonated, respectively. The slow phase of the photoreversal starts from a state with the release group deprotonated, and with the pK of Asp85 elevated, and is probably due to the restoration of the pK of Asp85 to its initial low value. This provides further evidence for coupling between the pKs of Asp85 and the release group and suggests that proton release is the first step in the reprotonation switch. At alkaline pH the amplitude of the electrical signal from the back photoreaction decreases with an apparent pK of 8, without a corresponding decrease in the amount of M. At neutral pH the movement of the positively charged guanidinium group of Arg82 from a position near the release group on the surface to Asp85 makes a substantial contribution to the electrical photoreversal amplitude. Above the pK of the release group in the unphotolysed state (approximately 8), Arg82 stays near the surface, leading to a corresponding signal reduction.