Orientation and dynamics of melittin in membranes of varying composition utilizing NBD fluorescence

Melittin is a cationic hemolytic peptide isolated from the European honey bee, Apis mellifera. The organization of membrane-bound melittin has earlier been shown to be dependent on the physical state and composition of membranes. In this study, we covalently labeled the N-terminal (Gly-1) and Lys-7 of melittin with an environment-sensitive fluorescent probe, the NBD group, to monitor the influence of negatively charged lipids and cholesterol on the organization and dynamics of membrane-bound melittin. Our results show that the NBD group of melittin labeled at its N-terminal end does not exhibit red edge excitation shift in DOPC and DOPC/DOPG membranes, whereas the NBD group of melittin labeled at Lys-7 exhibits REES of approximately 8 nm. This could be attributed to difference in membrane microenvironment experienced by the NBD groups in these analogs. Interestingly, the membrane environment of the NBD groups is sensitive to the presence of cholesterol, which is supported by time-resolved fluorescence measurements. Importantly, the orientation of melittin is found to be parallel to the membrane surface as determined by membrane penetration depth analysis using the parallax method in all cases. Our results constitute the first report to our knowledge describing the orientation of melittin in cholesterol-containing membranes. These results assume significance in the overall context of the role of membrane lipids in the orientation and function of membrane proteins and peptides.     

Interaction of melittin with membrane cholesterol: a fluorescence approach

We have monitored the organization and dynamics of the hemolytic peptide melittin in membranes containing cholesterol by utilizing the intrinsic fluorescence properties of its functionally important sole tryptophan residue and circular dichroism spectroscopy. The significance of this study is based on the fact that the natural target for melittin is the erythrocyte membrane, which contains high amounts of cholesterol. Our results show that the presence of cholesterol inhibits melittin-induced leakage of lipid vesicles and the extent of inhibition appears to be dependent on the concentration of membrane cholesterol. The presence of cholesterol is also shown to reduce binding of melittin to membranes. Our results show that fluorescence parameters such as intensity, emission maximum, and lifetime of membrane-bound melittin indicate a change in polarity in the immediate vicinity of the tryptophan residue probably due to increased water penetration in presence of cholesterol. This is supported by results from fluorescence quenching experiments using acrylamide as the quencher. Membrane penetration depth analysis by the parallax method shows that the melittin tryptophan is localized at a relatively shallow depth in membranes containing cholesterol. Analysis of energy transfer results using melittin tryptophan (donor) and dehydroergosterol (acceptor) indicates that dehydroergosterol is not randomly distributed and is preferentially localized around the tryptophan residue of membrane-bound melittin, even at the low concentrations used. Taken together, our results are relevant in understanding the interaction of melittin with membranes in general, and with cholesterol-containing membranes in particular, with possible relevance to its interaction with the erythrocyte membrane.     

Modulation of gramicidin channel conformation and organization by hydrophobic mismatch in saturated phosphatidylcholine bilayers

The matching of hydrophobic lengths of integral membrane proteins and the surrounding lipid bilayer is an important factor that influences both structure and function of integral membrane proteins. The ion channel gramicidin is known to be uniquely sensitive to membrane properties such as bilayer thickness and membrane mechanical properties. The functionally important carboxy terminal tryptophan residues of gramicidin display conformation-dependent fluorescence which can be used to monitor gramicidin conformations in membranes [S.S. Rawat, D.A. Kelkar, A. Chattopadhyay, Monitoring gramicidin conformations in membranes: a fluorescence approach, Biophys. J. 87 (2004) 831-843]. We have examined the effect of hydrophobic mismatch on the conformation and organization of gramicidin in saturated phosphatidylcholine bilayers of varying thickness utilizing the intrinsic conformation-dependent tryptophan fluorescence. Our results utilizing steady state and time-resolved fluorescence spectroscopic approaches, in combination with circular dichroism spectroscopy, show that gramicidin remains predominantly in the channel conformation and gramicidin tryptophans are at the membrane interfacial region over a range of mismatch conditions. Interestingly, gramicidin conformation shifts toward non-channel conformations in extremely thick gel phase membranes although it is not excluded from the membrane. In addition, experiments utilizing self quenching of tryptophan fluorescence indicate peptide aggregation in thicker gel phase membranes.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization

Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.     

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19 nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.     

Effect of ionic strength on folding and aggregation of the hemolytic peptide melittin in solution

Melittin is a cationic, amphipathic, hemolytic peptide composed of 26 amino acid residues. It is intrinsically fluorescent due to the presence of a single tryptophan residue, which has been shown to be crucial for its hemolytic activity. It undergoes a structural transition from a random coil monomer to an alpha-helical tetramer at high ionic strength. Although the aggregation behavior of melittin in solution is well characterized, dynamic information associated with the aggregation of melittin is lacking. In this paper, we have monitored the effect of ionic strength on the dynamics and aggregation behavior of melittin in aqueous solution by utilizing sensitive fluorescence approaches, which include the red edge excitation shift (REES) approach. Importantly, we demonstrate that REES is sensitive to the self-association of melittin induced by ionic strength. The change in environment experienced by melittin tryptophan(s) is supported by changes in fluorescence emission maximum, polarization, and lifetime. In addition, the accessibility of the tryptophan residue was probed by fluorescence quenching experiments using acrylamide and trichloroethanol as soluble and hydrophobic quenchers, respectively. Circular dichroism studies confirm the ionic strength-induced change in the secondary structure of melittin. Taken together, these results constitute the first report showing that REES could be used as a sensitive tool to monitor the aggregation behavior of melittin in particular and other proteins and peptides in general.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes

Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing differentially localized fluorescent membrane probes

Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using environment-sensitive and differentially localized fluorescent probes NBD-PE and NBD-cholesterol, utilizing wavelength-selective and time-resolved fluorescence measurements. The NBD group in NBD-PE is localized at the membrane interface while in NBD-cholesterol it is localized deeper in the membrane. Our results show that native hippocampal membranes offer considerable motional restriction as evidenced from red edge excitation shift of NBD probes. However, this effect progressively decreases with increasing cholesterol depletion in the case of NBD-cholesterol, possibly indicating a reduction in membrane heterogeneity. In contrast, REES of NBD-PE in hippocampal membranes does not show any significant change upon cholesterol depletion indicating relative lack of sensitivity of the membrane interface to cholesterol depletion. These observations are supported by changes in fluorescence polarization with cholesterol depletion. Taken together, these results imply that the deeper hydrocarbon region of the hippocampal membrane is more sensitive to changes in membrane organization and dynamics due to cholesterol depletion than the interfacial region. The motional restriction in native membranes is maintained even in the absence of proteins. The fluorescence lifetimes of both the NBD probes show slight reduction upon cholesterol depletion indicating a change in micro-environmental polarity possibly due to water penetration. These results are relevant in understanding the complex organization of hippocampal membranes and could have possible functional implications.     

Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

Influence of lipid chain unsaturation on membrane-bound melittin: a fluorescence approach

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. The organization of membrane-bound melittin is dependent on the physical state and composition of membranes. In particular, polyunsaturated lipids have been shown to modulate the membrane-disruptive action of melittin. Phospholipids with polyunsaturated acyl chains are known to modulate a number of physical properties of membranes and play an important role in regulating structure and function of membrane proteins. In this study, we have used melittin to address the influence of unsaturated lipids in modulating lipid-protein interactions. Our results show that fluorescence parameters such as intensity, emission maximum, polarization, lifetime and acrylamide quenching of melittin incorporated in membranes are dependent on the degree of unsaturation of lipids in membranes. Importantly, melittin in membranes composed of various unsaturated lipids shows red edge excitation shift (REES) implying that melittin is localized in a motionally restricted region in membranes. The extent of REES was found to increase drastically in membranes with increasing unsaturation, especially when the lipids contained more than two double bonds. In addition, increasing unsaturation in membranes causes a considerable change in the secondary structure of membrane-bound melittin. Taken together, our results assume significance in the overall context of the role of unsaturated lipids in membranes in the organization and function of membrane proteins and membrane-active peptides.     

Structural-dynamic properties of the tryptophan residue environment in melittin

The structural dynamics of the environment of the single tryptophan residue in melittin was studied by site-selective red-edge-excitation fluorescence spectroscopy. The dependence of the spectral shift on transition from excitation in a maximum (at 280 nm) to long-wavelength edge (305 nm) was studied as a function of temperature. It was shown, that for melittin at high ionic strength (tetramer), the three regions of temperature dependence of the red-edge effect are observed: retarded relaxation (up to +30 degrees C), relaxational changes of spectra (from +30 to +50 degrees C) and thermal changes of structure (above +50 degrees C). The dipolar-re-orientational relaxation time and activation energy of orientation motions in the environment of indolic ring in the tetrameric melittin structure were estimated. Extrapolation from relaxational region to room temperature results in relaxation time 40 ns. In monomeric melittin (at low ionic strength) the red-edge shift of spectra is absent. The distinct differences in character of thermal quenching of fluorescence between monomeric and tetrameric forms of melittin are observed. It follows, that the short-wave-length fluorescence shift on monomer-tetramer transition is due to both the reduction of polarity, and the increase in rigidity of tryptophan environment, the absence of relaxation motions at nanosecond times.     

Effect of structural transition of the host assembly on dynamics of an ion channel peptide: a fluorescence approach

Structural transition can be induced in charged micelles by increasing the ionic strength of the medium. We have monitored the organization and dynamics of the functionally important tryptophan residues of gramicidin in spherical and rod-shaped sodium dodecyl sulfate micelles utilizing a combination of wavelength-selective fluorescence and related fluorescence approaches. Our results show that tryptophans in gramicidin, present in the single-stranded beta(6.3) conformation, experience slow solvent relaxation giving rise to red edge excitation shift in spherical and rod-shaped micelles. In addition, changes in fluorescence polarization with increasing excitation or emission wavelength reinforce that the gramicidin tryptophans are localized in motionally restricted regions of these micelles. Fluorescence quenching experiments using acrylamide as a quencher of tryptophan fluorescence show that there is reduced water penetration in rod-shaped micelles. Taken together, we show that gramicidin conformation and dynamics is sensitive to the salt-induced structural transition in charged micelles. In addition, these results demonstrate that deformation of the host assembly could modulate protein conformation and dynamics.     

The gramicidin ion channel: A model membrane protein

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization, dynamics and function of membrane-spanning channels. In recent times, the availability of crystal structures of complex ion channels has challenged the role of gramicidin as a model membrane protein and ion channel. This review focuses on the suitability of gramicidin as a model membrane protein in general, and the information gained from gramicidin to understand lipid-protein interactions in particular. Special emphasis is given to the role and orientation of tryptophan residues in channel structure and function and recent spectroscopic approaches that have highlighted the organization and dynamics of the channel in membrane and membrane-mimetic media.     

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm^-1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm^-1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.     

Dynamics and heterogeneity of bovine hippocampal membranes: Role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

The component analysis of tryptophan fluorescence spectra of melittin during its oligomerization

The oligomerization of melittin with increasing ionic strength and protein concentration was investigated using the methods of decomposition of its tryptophan fluorescence spectra into "elementary" log-normal components. At high ionic strength (up to 2 M KCl), the emission spectra of tetrameric melittin are well described as the sum of two log-normal components, suggesting the presence of tryptophan residues in two sorts of environment with greatly differing polarity. Measurements of fluorescence spectra by iodide showed that these two spectral components possess different Stern-Volmer constants, that is, the tryptophans emitting them have different solvent accessibility, which does not correlate with the crystallographic structure of tetrameric melittin. Moreover, in the oligomerization transition induced by ionic strength, the tetrameric intermediate is formed, which has log-normal spectral components with relative contributions differing from those in 2 M KCl.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Dynamics of melittin in water and membranes as determined by fluorescence anisotropy decay.

Fluorescence anisotropy decay measurements were performed on melittin in water and in membranes of dimyristoylphosphatidylcholine. The fluorescence of the single tryptophan residue of melittin and of a pyrene label attached to melittin was detected. In water, the slowest relaxation process in the anisotropy decay occurs with a relaxation time of 1.5 or 5.5 ns in the case of low or high ionic strength and corresponds to rotational diffusion of monomeric or tetrameric melittin. Superimposed on this slow process are fast processes in the subnanosecond range reflecting fluctuations of the fluorophores relative to the polypeptide backbone. In membranes, the fast relaxation processes are not much altered. A slow process with a relaxation time of 35 ns is observed and assigned to orientational fluctuations of the melittin helices in membranes.     

Monitoring the looping up of acyl chain labeled NBD lipids in membranes as a function of membrane phase state

Lipids that are labeled with the NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group are widely used as fluorescent analogues of native lipids in biological and model membranes to monitor a variety of processes. The NBD group of acyl chain labeled NBD lipids is known to loop up to the membrane interface in fluid phase membranes. However, the organization of these lipids in gel phase membranes is not resolved. In this paper, we monitored the influence of the membrane phase state on the looping up behavior of acyl chain labeled NBD lipids utilizing red edge excitation shift (REES) and other sensitive fluorescence approaches. Interestingly, our REES results indicate that NBD group of lipids, which are labeled at the fatty acyl region, resides in the more hydrophobic region in gel phase membranes, and complete looping of the NBD group occurs only in the fluid phase. This is supported by other fluorescence parameters such as polarization and lifetime. Taken together, our results demonstrate that membrane packing, which depends on temperature and the phase state of the membrane, significantly affects the localization of acyl chain labeled NBD lipids. In view of the wide ranging use of NBD-labeled lipids in cell and membrane biology, these results could have potentially important implications in future studies involving these lipids as tracers.     

Effect of micellar charge on the conformation and dynamics of melittin

Electrostatic interactions play a crucial role in modulating and stabilizing molecular interactions in membranes and membrane-mimetic systems such as micelles. We have monitored the change in the conformation and dynamics of the cationic hemolytic peptide melittin bound to micelles of various charge types, utilizing fluorescence and circular dichroism (CD) spectroscopy. The sole tryptophan of melittin displays a red-edge excitation shift (REES) of 3-6 nm when bound to anionic, nonionic, and zwitterionic micelles. This suggests that melittin is localized in a restricted environment, probably in the interfacial region of the micelles, and this region offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state tryptophan in melittin. Further, the rotational mobility of melittin is considerably reduced in these micelles and is found to be dependent on the surface charge of micelles. Interestingly, our results show that melittin does not partition into cetyltrimethylammonium bromide (CTAB) micelles owing to electrostatic repulsion between melittin and CTAB micelles, both of which carry a positive charge. In addition, the fluorescence lifetime of melittin is modulated in micelles of different charge types. The lowest mean fluorescence lifetime is observed in the case of melittin bound to anionic sodium dodecyl sulfate (SDS) micelles. CD spectroscopy shows that micelles induce significant helicity to melittin, with maximum helicity being induced in the case of melittin bound to SDS micelles. Fluorescence quenching measurements using the neutral aqueous quencher acrylamide show differential accessibility of melittin in various types of micelles. Taken together, our results show that micellar surface charge can modulate the conformation and dynamics of melittin. These results could be relevant to understanding the role of the surface charge of membranes in the interaction of membrane-active, amphiphilic peptides with membranes.