Plastoquinol diffusion in linear photosynthetic electron transport

The diffusion of plastoquinol and its binding to the cytochrome bf complex, which occurs during linear photosynthetic electron transport and is analogous to reaction sequences found in most energy-converting membranes, has been studied in intact thylakoid membranes. The flash-induced electron transfer between the laterally separated photosystems II and photosystems I was measured by following the sigmoidal reduction kinetics of P-700⁺ after previous oxidation of the intersystem electron carriers. The amount of flash-induced plastoquinol produced at photosystem II was (a) reduced by inhibition with dichlorophenyl-dimethylurea and (b) increased by giving a second saturating flash. These signals were simulated by a new model which combines a deterministic simulation of reaction kinetics with a Monte Carlo approach to the diffusion of plastoquinol, taking into account the known structural features of the thylakoid membrane. The plastoquinol molecules were assumed to be oxidized by either a diffusion-limited or a nondiffusion-limited step in a collisional mechanism or after binding to the cytochrome bf complex. The model was able to account for the experimental observations with a nondiffusion-limited collisional mechanism or with a binding mechanism, giving minimum values for the diffusion coefficient of plastoquinol of 2 × 10^-8 cm²s^-1 and 3 × 10^-7 cm²s^-1, respectively.     

Diffusion in a Fluid Membrane with a Flexible Cortical Cytoskeleton

We calculate the influence of a flexible network of long-chain proteins, which is anchored to a fluid membrane, on protein diffusion in this membrane. This is a model for the cortical cytoskeleton and the lipid bilayer of the red blood cell, which we apply to predict the influence of the cytoskeleton on the diffusion coefficient of a mobile band 3 protein. Using the pressure field that the cytoskeleton exerts on the membrane, from the steric repulsion between the diffusing protein and the cytoskeletal filaments, we define a potential landscape for the diffusion within the bilayer. We study the changes to the diffusion coefficient on removal of one type of anchor proteins, e.g., in several hemolytic anemias, as well as for isotropic and anisotropic stretching of the cytoskeleton. We predict an overall increase of the diffusion for a smaller number of anchor proteins and increased diffusion for anisotropic stretching in the direction of the stretch, because of the decrease in the spatial frequency as well as in the height of the potential barriers.     

The membrane skeleton of erythrocytes. A percolation model.

The spectrin network on the cytoplasmic surface of the erythrocyte membrane is modeled as a triangular lattice of spectrin tetramers. This network obstructs lateral diffusion of proteins and provides mechanical reinforcement to the membrane. These effects are treated in a systematic and unified manner in terms of a percolation model. The diffusion coefficient is obtained as a function of the fraction of normal spectrin tetramers for both static and fluctuating barriers. The elasticity of the network is calculated as a function of the fraction of normal spectrin and the ratio of bending to stretching energies. For static barriers, elasticity and lateral diffusion are incompatible: if a network is connected enough to be elastic, it is connected enough to block long-range lateral diffusion. The elasticity and the force required for mechanical breakdown go to zero at the percolation threshold; experimental evidence suggests the existence of a stability threshold at or near the percolation threshold. The model is qualitatively applicable to other cells with membrane skeletons, such as epithelial cells, in which localization of membrane proteins is essential to differentiation.     

Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.     

Quantitative characterization of the lateral distribution of membrane proteins within the lipid bilayer.

The dependence of the lateral distribution of membrane proteins on the size, protein/lipoid molar ratio, and the magnitude of the interaction potentials has been investigated by computer modeling protein-lipid distributions with Monte Carlo calculations. These results have allowed us to develop a quantitative characterization of the distribution of membrane proteins and to correlate these distributions with experimental observables. The topological arrangement of protein domains, protein plus annular lipid domains, and free lipid domains is described in terms of radial distribution, pair connectedness, and cluster distribution functions. The radial distribution functions are used to measure the distribution of intermolecular distances between protein molecules, whereas the pair connectedness functions are used to estimate the physical extension of compositional domains. It is shown that, at characteristic protein/lipid molar ratios, previously isolated domains become connected, forming domain networks that extend over the entire membrane surface. These changes in the lateral connectivity of compositional domains are paralleled by changes in the calculated lateral diffusion coefficients and might have important implications for the regulation of diffusion controlled processes within the membrane.     

A kinetic, chromatographic method for studying protein hydrodynamic behavior

A chromatographic method based on "split-peak" behavior was described for the determination of the coefficient of mass transfer of proteins on small reversed-phase columns. The coefficient of mass transfer was found to be a linear function of the protein translational diffusion coefficient and inversely proportional to the square of the support particle diameter, as predicted by chromatographic theory. As an example of the practical application of this method for the measurement of protein diffusion coefficients, the denaturation of bovine serum albumin with decreasing solution pH was followed by measuring the change in the coefficient of mass transfer. A major advantage of this method was that the results were not affected by the interaction of the protein with the stationary phase.     

The plastoquinol–plastoquinone exchange mechanism in photosystem II: insight from molecular dynamics simulations

In the photosystem II (PSII) of oxygenic photosynthetic organisms, the reaction center (RC) core mediates the light-induced electron transfer leading to water splitting and production of reduced plastoquinone molecules. The reduction of plastoquinone to plastoquinol lowers PSII affinity for the latter and leads to its release. However, little is known about the role of protein dynamics in this process. Here, molecular dynamics simulations of the complete PSII complex embedded in a lipid bilayer have been used to investigate the plastoquinol release mechanism. A distinct dynamic behavior of PSII in the presence of plastoquinol is observed which, coupled to changes in charge distribution and electrostatic interactions, causes disruption of the interactions seen in the PSII–plastoquinone complex and leads to the “squeezing out” of plastoquinol from the binding pocket. Displacement of plastoquinol closes the second water channel, recently described in a 2.9 Å resolution PSII structure (Guskov et al. in Nat Struct Mol Biol 16:334–342, 2009), allowing to rule out the proposed “alternating” mechanism of plastoquinol–plastoquinone exchange, while giving support to the “single-channel” one. The performed simulations indicated a pivotal role of D₁-Ser264 in modulating the dynamics of the plastoquinone binding pocket and plastoquinol–plastoquinone exchange via its interaction with D₁-His252 residue. The effects of the disruption of this hydrogen bond network on the PSII redox reactions were experimentally assessed in the D₁ site-directed mutant Ser264Lys.     

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)¹ was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a "slope of slopes" plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.     

Reaction pattern of Photosystem II: oxidative water cleavage and protein flexibility

This short communication addresses three topics of photosynthetic water cleavage in Photosystem II (PS II): (a) effect of protonation in the acidic range on the extent of the ‘fast’ ns kinetics of P680^+· reduction by Y_Z, (b) mechanism of O–O bond formation and (c) role of protein flexibility in the functional integrity of PS II. Based on measurements of light-induced absorption changes and quasielastic neutron scattering in combination with mechanistic considerations, evidence is presented for the protein acting as a functionally active constituent of the water cleavage machinery, in particular, for directed local proton transfer. A specific flexibility emerging above a threshold of about 230 K is an indispensable prerequisite for oxygen evolution and plastoquinol formation.     

Flexibility in photosynthetic electron transport: the physiological role of plastoquinol terminal oxidase (PTOX)

Oxygenic photosynthesis depends on a highly conserved electron transport system, which must be particularly dynamic in its response to environmental and physiological changes, in order to avoid an excess of excitation energy and subsequent oxidative damage. Apart from cyclic electron flow around PSII and around PSI, several alternative electron transport pathways exist including a plastoquinol terminal oxidase (PTOX) that mediates electron flow from plastoquinol to O(2). The existence of PTOX was first hypothesized in 1982 and this was verified years later based on the discovery of a non-heme, di-iron carboxylate protein localized to thylakoid membranes that displayed sequence similarity to the mitochondrial alternative oxidase. The absence of this protein renders higher plants susceptible to excitation pressure dependant variegation combined with impaired carotenoid synthesis. Chloroplasts, as well as other plastids (i.e. etioplasts, amyloplasts and chromoplasts), fail to assemble organized internal membrane structures correctly, when exposed to high excitation pressure early in development. While the role of PTOX in plastid development is established, its physiological role under stress conditions remains equivocal and we postulate that it serves as an alternative electron sink under conditions where the acceptor side of PSI is limited. The aim of this review is to provide an overview of the past achievements in this field and to offer directions for future investigative efforts. Plastoquinol terminal oxidase (PTOX) is involved in an alternative electron transport pathway that mediates electron flow from plastoquinol to O(2). This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Protein lateral movement in lipid bilayers. Stimulation studies of its dependence upon protein concentration

A number of mechanisms have been proposed to account for the decrease in protein lateral diffusion coefficients in a lipid bilayer membrane, as the concentration of proteins is increased. One such mechanism is the steric hindrance (via, say, a hard-core repulsion) to the lateral movement of a protein due to the proximity of other proteins. Here a model is presented to study this effect alone. It is argued that the model will overestimate the effect being studied. The results of computer simulations show that such a mechanism will decrease the lateral diffusion coefficient by less than a factor of 20 below the zero-concentration limit, even when up to 81.7% of the bilayer surface is composed of integral proteins. This result supports the opinion (Kell, D.B. (1984) Trends Biochem. Sci. 9, 379) that such a mechanism cannot account for a decrease in the lateral diffusion coefficient by two or three orders of magnitude.     

Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion

Analysis of particle trajectories in images obtained by fluorescence microscopy reveals biophysical properties such as diffusion coefficient or rates of association and dissociation. Particle tracking and lifetime measurement is often limited by noise, large mobilities, image inhomogeneities, and path crossings. We present Speckle TrackerJ, a tool that addresses some of these challenges using computer-assisted techniques for finding positions and tracking particles in different situations. A dynamic user interface assists in the creation, editing, and refining of particle tracks. The following are results from application of this program: 1), Tracking single molecule diffusion in simulated images. The shape of the diffusing marker on the image changes from speckle to cloud, depending on the relationship of the diffusion coefficient to the camera exposure time. We use these images to illustrate the range of diffusion coefficients that can be measured. 2), We used the program to measure the diffusion coefficient of capping proteins in the lamellipodium. We found values ∼0.5 μm²/s, suggesting capping protein association with protein complexes or the membrane. 3), We demonstrate efficient measuring of appearance and disappearance of EGFP-actin speckles within the lamellipodium of motile cells that indicate actin monomer incorporation into the actin filament network. 4), We marked appearance and disappearance events of fluorescently labeled vesicles to supported lipid bilayers and tracked single lipids from the fused vesicle on the bilayer. This is the first time, to our knowledge, that vesicle fusion has been detected with single molecule sensitivity and the program allowed us to perform a quantitative analysis. 5), By discriminating between undocking and fusion events, dwell times for vesicle fusion after vesicle docking to membranes can be measured.     

A Highlights from MBoC Selection: Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface.     

Investigating the production of foreign membrane proteins in tobacco chloroplasts: expression of an algal plastid terminal oxidase

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5'UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast.     

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.     

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.     

Time-dependent absorption anisotropy and rotational diffusion of proteins in membranes.

The decay of flash-induced absorption anisotropy, r(t), of a chromophore in a membrane protein is closely correlated with rotational diffusion of the protein in the membrane. We develop a theory of time-dependent absorption anisotropy which is applicable to both linear chromophores and planar chromophores which have two different absorption moments at right angles to one another. The theory treats two types of rotational diffusion of membrane proteins: one is rotation of the whole protein about the normal to the plane of the membrane, and the other is restricted wobbling of the whole or part of the protein molecule. In the former case, r(t) is determined by a rotational diffusion coefficient and an angle between the absorption moment(s) and the normal to the plane of the membrane. Rotation of rigid transmembrane proteins can be described by this treatment. In the latter case, r(t) is characterized by a wobbling diffusion coefficient and the degree of orientational constraint. This treatment may be applicable to independent wobbling of the hydrophilic part of membrane proteins. We further show that, for linear and circularly degenerate chromophores, the effect of the excitation flash intensity on r(t) can be accounted for by a constant scaling factor.     

A pulsed field gradient NMR study of the aggregation and hydration of parvalbumin

Pulsed field gradient NMR is a convenient alternative to traditional methods for measuring diffusion of biological macromolecules. In the present study, pulsed field gradient NMR was used to study the effects of calcium binding and hydration on carp parvalbumin. Carp parvalbumin is known to undergo large changes in tertiary structure with calcium loading. The diffusion coefficient is a sensitive guide to changes in molecular shape and in the present study the large changes in tertiary structure were clearly reflected in the measured diffusion coefficient upon calcium loading. The (monomeric) calcium-loaded form had a diffusion coefficient of 1.4 x 10(-10) m(2) s(-1) at 298 K, which conforms with the structure being a nearly spherical prolate ellipsoid from X-ray studies. The calcium-free form had a significantly lower diffusion coefficient of 1.1 x 10(-10) m(2) s(-1). The simplest explanation consistent with the change in diffusion coefficient is that the parvalbumin molecules form dimers upon the removal of Ca(2+) at the protein concentration studied (1 mM).