Organization of protochlorophyllide oxidoreductase in prolamellar bodies isolated from etiolated carotenoid-deficient wheat leaves as revealed by fluorescence probes

Carotenoid importance for membrane organization of NADPH protochlorophyllide oxidoreductase (POR) was studied by comparing interaction of two membrane fluorescent probes with proteins in prolamellar bodies isolated from norflurazon-treated wheat plants (cdPLBs) to those isolated form plants with normal carotenoid amount (oPLBs). The tryptophan fluorescence quenching by 1-anilino-8-naphthalene sulfonate (attached to the surface of membrane lipid phase) and pyrene (situated deep into the fatty acid region of membrane lipids) was used to locate the position of POR molecules toward lipid phase, to analyze their supramolecular organization and the light-induced structural transitions. Our results showed that the pigment-protein complexes of cdPLBs were larger than those of oPLBs. Upon flash irradiation the aggregates of both types of PLB dissociated into smaller units but in cdPLBs this process was accompanied by reorientation of the POR molecules closer to the lipid surface and/or dissociation from the lipids. These results revealed that carotenoid deficiency led to a looser attachment of POR to the lipid phase and its early (in comparison with oPLBs) dissociation from the membranes during the light-induced transformation of cdPLBs. This might be one of the reasons for the inability of carotenoid-deficient plants to form functional plastids.     

Lipid-protein interaction in reconstituted cytochrome c oxidase/phospholipid membranes.

Deuterium and 31P nuclear magnetic resonance have been employed in an investigation of the effect of cytochrome c oxidase (EC 1.9.3.1) on the structure of lecithin bilayers. Cytochrome c oxidase was isolated from beef heart mitochondria in lipid-free form and reconstituted as a functional enzyme in bilayers composed of synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Two separate reconstitution experiments were performed in which the lipid was selectively deuterated either at the C-5' or at the C-14' segment of the palmitic acyl chain. The phospholipid-to-protein ratio of both reconstituted complexes was 0.74 (mg/mg), corresponding to about 200 molecules lipid per molecule cytochrome c oxidase. The deuterium quadrupole splitting deltanuQ, and the phosphorus chemical shielding anisotropy, deltasigma, of the cytochrome c oxidase-phospholipid recombinants were measured as a function of temperature and compared to the results obtained for the pure lipid membrane without protein for the pure lipid membrane without protein. deltanuQ and deltasigma are highly sensitive to the structural organization of the lipid membrane and these measurements demonstrate that the incorporation of cytochrome c oxidase into phosphatidylcholine bilayers leads to a more disordered conformational state of the lipids. This result can be explained by a rapid exchange between lipids in direct contact with hydrophobic protein and those further away from it (exchange rate greater than 10(4) Hz). The irregular protein surface is sensed by all lipid molecules and induces a more disordered bilayer structure. In contrast to previous interpretations, our measurements do not suggest a special type of boundary lipid.     

Assembly of NADPH:protochlorophyllide oxidoreductase complex is needed for effective greening of barley seedlings

NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR) is the key enzyme in the light-induced greening of higher plants. A unique light-harvesting POR:Pchlide complexes (LHPP) has been found in barley etioplasts, but not in other plant species. Why PORs from barley, but not from other plants, can form LHPP? And its function is not well understood. We modeled the barley and Arabidopsis POR proteins and compared molecular surface. The results confirm the idea that barley PORA can form a five-unit oligomer that interacts with a single PORB. Chemical treatment experiments indicated that POR complex may be formed by dithiol oxidation of cysteines of two adjacent proteins. We further showed that LHPP assembly was needed for barley POR functions and seedling greening. On the contrary, Arabidopsis POR proteins only formed dimers, which were not related to the functions or the greening. Finally, POR complex assembly (including LHPP and POR dimers) did not affect the formation of prolamellar bodies (PLBs) that function for efficient capture of light energy for photo conversion in etioplasts.     

Effect of Periodic Heat Shock on the Inner Membrane System of Etioplasts

Etiolated barley (Hordeum vulgare L.) seedlings were treated with heat shock (HS). The heat treatment was conducted daily for 1 h at 40°C over 6 days and led to shortening of leaves and coleoptiles, an increase in the etioplast volume and prothylakoid length, and to a decrease in the size of paracrystalline prolamellar bodies (PLB). As a result of HS treatment, stimulation of carotenoid and protochlorophyllide (Pchlide) synthesis as well as an increase in the relative content of the Pchlide short-wavelength form (Pchlide₆₃₀) were observed in the leaf tissue of seven-day-old seedlings 12 h after the last HS treatment. HS had no effect on the overall amount of Pchlide-oxidoreductase (POR) in leaves and PLB membranes and did not suppress the Pchlide photoreduction in vivo. PLB membranes, isolated from the HS-treated seedlings, possessed a higher Pchlide and carotenoid content as calculated on total protein basis. These membranes showed more intense protein fluorescence than PLB from untreated plants, whereas hydrophobicity of the microenvironment of the fluorescent amino-acid residues remained unchanged. Studies using pyrene (lipophilic fluorescent probe emitted in Pchlide and carotenoid absorption bands) showed that HS increases the fluidity of membrane lipids in PLB membranes and that the pigments accumulated in these membranes are located in the region of lipid–protein contact site. The results are discussed in relation to the adaptive role of protein–protein and pigment–protein–lipid interactions in etioplast membranes under stress.     

Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-L-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-L-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry. At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 A with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 A2 to a limiting value of 100 A2. On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93,000. This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles.     

Studies on canthaxanthin in lipid membranes

Polar carotenoid pigment - canthaxanthin - has been found to interfere with the organization of biological membranes, in particular of the retina membranes of an eye of primates. The organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) and egg yolk phosphatidylcholine containing canthaxanthin was studied by means of several techniques including: electronic absorption spectroscopy, linear dichroism, X-ray diffractometry, ¹H-NMR spectroscopy and FTIR spectroscopy. It appears that canthaxanthin present in the lipid membranes at relatively low concentration (below 1 mol% with respect to lipid) modifies significantly physical properties of the membranes. In particular, canthaxanthin (i) exerts restrictions to the segmental molecular motion of lipid molecules both in the headgroup region and in the hydrophobic core of the bilayer, (ii) promotes extended conformation of alkyl lipid chains, (iii) modifies the surface of the lipid membranes (in particular in the gel state, L_β´) and promotes the aggregation of lipid vesicles. It is concluded that canthaxanthin incorporated into lipid membranes is distributed among two pools: one spanning the lipid bilayer roughly perpendicularly to the surface of the membrane and one parallel to the membrane, localized in the headgroup region. The population of the horizontal fraction increases with the increase in the concentration of the pigment in the lipid phase. Such a conclusion is supported by the linear dichroism analysis of the oriented lipid multibilayers containing canthaxanthin: The mean angle between the dipole transition moment and the axis normal to the plane of the membrane was determined as 20 ± 3° at 0.5 mol% and 47 ± 3° at 2 mol% canthaxanthin. The analysis of the absorption spectra of canthaxanthin in the lipid phase and ¹H-NMR spectra of lipids point to the exceptionally low aggregation threshold of the pigment in the membrane environment (∼1 mol%). All results demonstrate a very strong modifying effect of canthaxanthin with respect to the dynamic and structural properties of lipid membranes.     

Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance

Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase. Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase). Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains. A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range. Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases. The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains. These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins. It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.     

Spin-labeling study of membranes in wheat embryo axes. 1. Partitioning of doxyl stearates into the lipid domains

The interaction of lipid soluble spin labels with wheat embryo axes has been investigated to obtain insight into the structural organization of lipid domains in embryo cell membranes, using conventional electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopy. Stearic acid spin labels (n-SASL) and their methylated derivatives (n-MeSASL), labelled at different positions of their doxyl group (n=5, 12 and 16), were used to probe the ordering and molecular mobility in different regions of the lipid moiety of axis cell membranes. The ordering and local polarity in relation to the position of the doxyl group along the hydrocarbon chain of SASL, determined over the temperature range from -50 to +20 degrees C, are typical for biological and model lipid membranes, but essentially differ from those in seed oil droplets. Positional profiles for ST-EPR spectra show that the flexibility profile along the lipid hydrocarbon chain does exist even at low temperatures, when most of the membrane lipids are in solid state (gel phase). The ordering of the SASL nitroxide radical in the membrane surface region is essentially higher than that in the depth of the membrane. The doxyl groups of MeSASLs are less ordered (even at low temperatures) than those of the corresponding SASLs, indicating that the MeSASLs are located in the bulk of membrane lipids rather than in the protein boundary lipids. The analysis of the profiles of EPR and ST-EPR spectral parameters allows us to conclude that the vast majority of SASL and MeSASL molecules accumulated in embryo axes is located in the cell membranes rather than in the interior of the oil bodies. The preferential partitioning of the doxyl stearates into membranes demonstrates the potential of the EPR spin-labelling technique for the in situ study of membrane behavior in seeds of different hydration levels.     

Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis(phosphate) in water

The molecular organization of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis-(phosphate)/water systems is investigated over a wide range of lipid concentrations using X-ray diffraction, calorimetry, analytical ultracentrifugation, densitometry and viscometry.At high lipid concentrations, the lipid molecules are found to form a lamellar phase. The repeat distance increases from 60 to 120 Å with increasing water content to 70 wt% and the surface area per lipid molecule increases from 41.7 Ų to a limiting value of 100 Ų.On the other hand, at very low lipid concentrations the molecules are found to form not vesicles but micelles, the total molecular weight of which takes a value of 93 000.This finding revises the prevalent view that lipids containing two (or more) hydrocarbon chains form extended bilayers or vesicles, whereas single chained lipids form micelles. (Tanford, C.(1972) J. Phys. Chem. 76, 3020–3024).     

Monitoring Shifts in the Conformation Equilibrium of the Membrane Protein Cytochrome P450 Reductase (POR) in Nanodiscs

Nanodiscs are self-assembled ∼50-nm² patches of lipid bilayers stabilized by amphipathic belt proteins. We demonstrate that a well ordered dense film of nanodiscs serves for non-destructive, label-free studies of isolated membrane proteins in a native like environment using neutron reflectometry (NR). This method exceeds studies of membrane proteins in vesicle or supported lipid bilayer because membrane proteins can be selectively adsorbed with controlled orientation. As a proof of concept, the mechanism of action of the membrane-anchored cytochrome P450 reductase (POR) is studied here. This enzyme is responsible for catalyzing the transfer of electrons from NADPH to cytochrome P450s and thus is a key enzyme in the biosynthesis of numerous primary and secondary metabolites in plants. Neutron reflectometry shows a coexistence of two different POR conformations, a compact and an extended form with a thickness of 44 and 79 Å, respectively. Upon complete reduction by NADPH, the conformational equilibrium shifts toward the compact form protecting the reduced FMN cofactor from engaging in unspecific electron transfer reaction.     

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.     

Thermotropic lipid phase transition and the behavior of hydrolytic enzymes in the kidney cortex brush border membrane

Functional interactions of lipids and proteins were examined in brush-border membranes isolated from the kidney cortex by studying the temperature dependence of the hydrolytic enzyme activities. A close relationship was observed for the membrane proteins and the thermotropic lipid phase transitions. Three lines of evidences were provided for such dependence: a) Arrhenius relationship of the membrane-bound enzyme activities, and the effect of temperature in native and partially delipidated membranes, b) differential scanning calorimetric study of the membrane lipid phase transitions in the native and delipidated membranes, multilamellar vesicles prepared from the membrane extracted lipids, and in vesicles from dimyristoyl phosphatidylcholine, and c) the excimer (dimer)-formation studies of the membrane extrinsic fluorescent probe, pyrene, and the resultant membrane microviscosity. The brush-border membranes were partially delipidated with BuOH and 2,2,2-trifluoroethanol. The functional interactions of the delipidated membranes, which were greatly lost on lipid removal, were largely restored by the addition of exogenous lipids in the reconstitution process, which indicate the critical dependence of the membrane integral proteins on the neighboring lipid molecules in the bulk lipid phase.     

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer.

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.     

Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. I. Structure of the molecular complex

This paper reports on a simulation of a gramicidin channel inserted into a fluid phase DMPC bilayer with 100 lipid molecules. Two lipid molecules per leaflet were removed to insert the gramicidin, so the resulting preparation had 96 lipid molecules and 3209 water molecules. Constant surface tension boundary conditions were employed. Like previous simulations with a lower lipid/gramicidin ratio (Woolf, T. B., and B. Roux. 1996. Proteins: Struct., Funct., Genet. 24:92-114), it is found that tryptophan-water hydrogen bonds are more common than tryptophan-phospholipid hydrogen bonds. However, one of the tryptophan NH groups entered into an unusually long-lived hydrogen bonding pattern with two glycerol oxygens of one of the phospholipid molecules. Comparisons were made between the behavior of the lipids adjacent to the channel with those farther away. It was found that hydrocarbon chains of lipids adjacent to the channel had higher-order parameters than those farther away. The thickness of the lipid bilayer immediately adjacent to the channel was greater than it was farther away. In general, the lipids adjacent to the membrane had similar orientations to those seen by Woolf and Roux, while those farther away had similar orientations to those pertaining before the insertion of the gramicidin. A corollary to this observation is that the thickness of the hydrocarbon region adjacent to the gramicidin was much thicker than what other studies have identified as the "hydrophobic length" of the gramicidin channel.     

Biomimetic lipid/polymer colorimetric membranes: molecular and cooperative properties

Characterization of membranes and of biological processes occurring within membranes is essential for understanding fundamental cellular behavior. Here we present a detailed biophysical study of a recently developed colorimetric biomimetic membrane assembly constructed from physiological lipid molecules and conjugated polydiacetylene. Various analytical techniques have been applied to characterize the organization of the lipid components in the chromatic vesicles and their contributions to the observed blue-to-red color transitions. Experiments reveal that both the polymerized units as well as the lipids exhibit microscopic phases and form domains whose properties and bilayer organization are interdependent. These domains are interspersed within mixed lipid/polymer vesicles that have a size distribution different from those of aggregates of the individual molecular constituents. The finding that fluidity changes induced within the lipid domains are correlated with the chromatic transitions demonstrates that the colorimetric platform can be used to evaluate the effects of individual molecular components, such as negatively charged lipids and cholesterol, upon membrane fluidity and thermal stability.     

Reduced graphene oxide directed self-assembly of phospholipid monolayers in liquid and gel phases

The response of cell membranes to the local physical environment significantly determines many biological processes and the practical applications of biomaterials. A better understanding of the dynamic assembly and environmental response of lipid membranes can help understand these processes and design novel nanomaterials for biomedical applications. The present work demonstrates the directed assembly of lipid monolayers, in both liquid and gel phases, on the surface of a monolayered reduced graphene oxide (rGO). The results from atomic force microscopy indicate that the hydrophobic aromatic plane and the defect holes due to reduction of GO sheets, along with the phase state and planar surface pressure of lipids, corporately determine the morphology and lateral structure of the assembled lipid monolayers. The DOPC molecules, in liquid phase, probably spread over the rGO surface with their tails associating closely with the hydrophobic aromatic plane, and accumulate to form circles of high area surrounding the defect holes on rGO sheets. However, the DPPC molecules, in gel phase, prefer to form a layer of continuous membrane covering the whole rGO sheet including defect holes. The strong association between rGO sheets and lipid tails further influences the melting behavior of lipids. This work reveals a dramatic effect of the local structure and surface property of rGO sheets on the substrate-directed assembly and subsequent phase behavior of the supported lipid membranes.     

Interaction among molecules in mixtures of ceramide/stearic acid,ceramide/cholesterol and ceramide/stearic acid/cholesterol

To elucidate the interaction among the molecules which constitute intercellular lipids of stratum corneum, the phase diagrams in the binary mixtures of N-octadecanoyl-phytosphingosine (CER)/stearic acid (SA) and CER/cholesterol (CHOL) were studied by differential scanning calorimetry and by small- and wide-angle X-ray diffraction. These phase diagrams are mostly expressed by a eutectic type one. However, from their detailed analyses, it was revealed that in the phase diagram of CER/SA a new solid structure is formed just above the eutectic temperature. The lamellar spacing of the new structure is nearly equal to the length given by the sum of the two molecules of CER and/or SA, that is, in the lipid bilayer the hydrocarbon chains of CER and SA lie almost perpendicular to the lipid bilayer surface and the two kinds of molecules distribute homogeneously. On the other hand, in the binary mixture of CER/CHOL, CHOL molecules are apt to be isolated from the mixture. In a ternary mixture composed of equimolar lipids of CER, CHOL and SA, it was found that a pseudo-hexagonal structure takes place even in the solid state. This fact indicates that the three components are miscible and the hydrocarbon chains lie perpendicular to the lipid bilayer surface. We can draw the conclusion that the multi-component mixtures containing ceramide are apt to form the lamellar structure where even in the solid state the hydrocarbon chains lie perpendicular to the lipid bilayer surface and the components with hydrocarbon chains distribute homogeneously.     

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Increases in lutein/carotenoid ratios lead to decreased thylakoid fluidity and hamper grana folding due to carotenoid-dependent changes in both photosynthetic complexes and lipid matrix organization.     

Single-Molecule Fluorescence Studies of a PH Domain: New Insights into the Membrane Docking Reaction

Proteins containing membrane targeting domains play essential roles in many cellular signaling pathways. However, important features of the membrane-bound state are invisible to bulk methods, thereby hindering mechanistic analysis of membrane targeting reactions. Here we use total internal reflection fluorescence microscopy (TIRFM), combined with single particle tracking, to probe the membrane docking mechanism of a representative pleckstrin homology (PH) domain isolated from the general receptor for phosphoinositides, isoform 1 (GRP1). The findings show three previously undescribed features of GRP1 PH domain docking to membranes containing its rare target lipid, phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P₃]. First, analysis of surface diffusion kinetics on supported lipid bilayers shows that in the absence of other anionic lipids, the PI(3,4,5)P₃-bound protein exhibits the same diffusion constant as a single lipid molecule. Second, the binding of the anionic lipid phosphatidylserine to a previously unidentified secondary binding site slows both diffusion and dissociation kinetics. Third, TIRFM enables direct observation of rare events in which dissociation from the membrane surface is followed by transient diffusion through solution and rapid rebinding to a nearby, membrane-associated target lipid. Overall, this study shows that in vitro single-molecule TIRFM provides a new window into the molecular mechanisms of membrane docking reactions.