Semisynthesis and folding of the potassium channel KcsA

In this contribution we describe the semisynthesis of the potassium channel, KcsA. A truncated form of KcsA, comprising the first 125 amino acids of the 160-amino acid protein, was synthesized using expressed protein ligation. This truncated form corresponds to the entire membrane-spanning region of the protein and is similar to the construct previously used in crystallographic studies on the KcsA protein. The ligation reaction was carried out using an N-terminal recombinant peptide alpha-thioester, corresponding to residues 1-73 of KcsA, and a synthetic C-terminal peptide corresponding to residues 74-125. Chemical synthesis of the C-peptide was accomplished by optimized Boc-SPPS techniques. A dual fusion strategy, involving glutathione-S-transferase (GST) and the GyrA intein, was developed for recombinant expression of the N-peptide alpha-thioester. The fusion protein, expressed in the insoluble form as inclusion bodies, was refolded and then cleaved successively to remove the GST tag and the intein, thereby releasing the N-peptide alpha-thioester. Following chemical ligation, the KcsA polypeptide was folded into the tetrameric state by incorporation into lipid vesicles. The correctness of the folded state was verified by the ability of the KcsA tetramer to bind to agitoxin-2. To our knowledge, this work represents the first reported semisynthesis of a polytopic membrane protein and highlights the potential application of native chemical ligation and expressed protein ligation for the (semi)synthesis of integral membrane proteins.     

Kinetic modeling of ion conduction in KcsA potassium channel

KcsA constitutes a potassium channel of known structure that shows both high conduction rates and selectivity among monovalent cations. A kinetic model for ion conduction through this channel that assumes rapid ion transport within the filter has recently been presented by Nelson. In a recent, brief communication, we used the model to provide preliminary explanations to the experimental current-voltage J-V and conductance-concentration g-S curves obtained for a series of monovalent ions (K(+),Tl(+), and Rb(+)). We did not assume rapid ion transport in the calculations, since ion transport within the selectivity filter could be rate limiting for ions other than native K(+). This previous work is now significantly extended to the following experimental problems. First, the outward rectification of the J-V curves in K(+) symmetrical solutions is analyzed using a generalized kinetic model. Second, the J-V and g-S curves for NH(4) (+) are obtained and compared with those of other ions (the NH(4) (+) J-V curve is qualitatively different from those of Rb(+) and Tl(+)). Third, the effects of Na(+) block on K(+) and Rb(+) currents through single KcsA channels are studied and the different blocking behavior is related to the values of the translocation rate constants characteristic of ion transport within the filter. Finally, the significantly decreased K(+) conductance caused by mutation of the wild-type channel is also explained in terms of this rate constant. In order to keep the number of model parameters to a minimum, we do not allow the electrical distance (an empirical parameter of kinetic models that controls the exponential voltage dependence of the dissociation rate) to vary with the ionic species. Without introducing the relatively high number of adjustable parameters of more comprehensive site-based models, we show that ion association to the filter is rate controlling at low concentrations, but ion dissociation from the filter and ion transport within the filter could limit conduction at high concentration. Although some experimental data from other authors were included to allow qualitative comparison with model calculations, the absolute values of the effective rate constants obtained are only tentative. However, the relative changes in these constants needed to explain qualitatively the experiments should be of significance.     

Shape changes and vesicle fission of giant unilamellar vesicles of liquid-ordered phase membrane induced by lysophosphatidylcholine

Liquid-ordered phase (lo phase) of lipid membranes has properties that are intermediate between those of liquid-crystalline phase and those of gel phase and has attracted much attention in both biological and biophysical aspects. Rafts in the lo phase in biomembranes play important roles in cell function of mammalian cells such as signal transduction. In this report, we have prepared giant unilamellar vesicles (GUVs) of lipid membranes in the lo phase and investigated their physical properties using phase-contrast microscopy and fluorescence microscopy. GUVs of dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol membranes and also GUVs of sphingomyelin (SM)/cholesterol membranes in the lo phase in water were formed at 20-37 degrees C successfully, when these membranes contained >/=30 mol % cholesterol. The diameters of GUVs of DPPC/cholesterol and SM/cholesterol membranes did not change from 50 to 28 degrees C, supporting that the membranes of these GUVs were in the lo phase. To elucidate the interaction of a substance with a long hydrocarbon chain with the lo phase membrane, we investigated the interaction of low concentrations (less than critical micelle concentration) of lysophosphatidylcholine (lyso-PC) with DPPC/cholesterol GUVs and SM/cholesterol GUVs in the lo phase. We found that lyso-PC induced several shape changes and vesicle fission of these GUVs above their threshold concentrations in water. The analysis of these shape changes indicates that lyso-PC can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. Threshold concentrations of lyso-PC in water to induce the shape changes and vesicle fission increased greatly with a decrease in chain length of lyso-PC. Thermodynamic analysis of this result indicates that shape changes and vesicle fission occur at threshold concentrations of lyso-PC in the membrane. These new findings on GUVs of the lo phase membranes indicate that substances with a long hydrocarbon chain such as lyso-PC can enter into the lo phase membrane and also the raft in the cell membrane. We have also proposed a mechanism for the lyso-PC-induced vesicle fission of GUVs.     

Role of water molecules in the KcsA protein channel by molecular dynamics calculations

Molecular dynamics simulations supported by electrostatic calculations have been conducted on the KcsA channel to determine the role of water molecules in the pore. Starting from the X-ray structure of the KcsA channel in its closed state at 2.0 angstroms resolution, the opening of the pore towards a conformation built on the basis of EPR results is studied. We show that water molecules act as a structural element for the K+ ions inside the filter and the hydrophobic cavity of the channel. In the filter, water tends to enhance the depth of the wells occupied by the K+ ions, while in the cavity there is a strong correlation between the water molecules and the cavity ion. As a consequence, the protein remains very stable in the presence of three K+ ions in the selectivity filter and one in the cavity. The analysis of the dynamics of water molecules in the cavity reveals preferred orientations of the dipoles along the pore axis, and a correlated behavior between this dipole orientation and the displacement of the K+ ion during the gating process.     

The effect of membrane fluidity on FRET parameters: an energy transfer study inside small unilamellar vesicle

The fluorescence resonance energy transfer (FRET) in a lipid bilayer system containing two different donors and one common acceptor at below and above transition temperature has been studied and all the FRET parameters are analyzed using steady state and time-resolved fluorescence spectroscopy. Using dynamic light scattering measurement, we have followed the process of preparation of small unilamellar vesicles, and by following the FRET parameters of C-153-Rh6G and C-151-Rh6G pairs inside SUVs at 16 °C and 33 °C (T(m) = 23.9 °C) we have noticed that there is greater effect of temperature on the FRET parameters in case of the C-153-Rh6G pair than that of the C-151-Rh6G pair. Finally we have concluded that this difference is due to their different location inside the lipid bilayer in which fluidity of the long alkyl chain markedly affects the FRET parameters for C-153-Rh6G pair embedded inside a small unilamellar vesicle of size 20-50 nm.     

Role of fluctuations in a snug-fit mechanism of KcsA channel selectivity

The thermodynamic exclusion of Na+ relative to K+ in potassium channels is examined by calculating the distribution of binding energies for Na+ and K+ in a model of the selectivity filter of the KcsA potassium channel. These distributions are observed to take a surprisingly simple form: Gaussian with a slight positive skewness that is insignificant in the present context. Complications that might be anticipated from these distributions are not problematic here. Na+ occupies the filter with a mean binding energy substantially lower than that of K+. The difference is comparable to the difference in hydration free energies of Na+ and K+ in bulk aqueous solution. Thus, the average energies of binding to the filter do not discriminate Na+ from K+ when measured from a baseline of the difference in bulk hydration free energies. The strong binding of Na+ constricts the filter, consistent with a negative partial molar volume of Na+ in water in contrast with a positive partial molar volume of K+ in water. Discrimination in favor of K+)can be attributed to the scarcity of favorable binding configurations for Na+ compared to K+. That relative scarcity is quantified as enhanced binding energy fluctuations, which reflects both the energetically stronger binding of Na+ and the constriction of the filter induced by Na+ binding.     

Lipid diffusion from single molecules of a labeled protein undergoing dynamic association with giant unilamellar vesicles and supported bilayers

It is demonstrated that single-molecule tracking of a fluorescently labeled protein undergoing transient binding to model membranes presents a useful method of obtaining fluid properties. The labeled ACBP protein was tracked during its binding to free-standing giant unilamellar vesicles (GUVs) and supported bilayers prepared from the GUVs in the same environment. The analysis of images that are blurred as a result of fast probe diffusion was discussed. An examination of the lateral diffusion trajectories revealed a homogeneous diffusion on the top segments of the GUVs with D = 6.9 +/- 0.3 microm(2)/s. The supported bilayer experiments revealed two diffusion processes, one with Df = 3.1 +/- 0.4 microm(2)/s and the other with Ds = 0.078 +/- 0.001 microm(2)/s. The 2-fold difference in the lipid bilayer mobility for the free-standing and fast components in the supported bilayers is attributed to the known effect of frictional coupling with the solid support. The slow mobile fraction in the bilayer is suggested to be associated with the migration of pore-like structures, originating from the interaction of the membrane with the glass support.     

Surface thermodynamic properties of monolayers versus reconstitution of a membrane protein in solid-supported bilayers

Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air-water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix.     

Controlled hydrogel formation in the internal compartment of giant unilamellar vesicles

The introduction of poly(ethylene dioxythiophene) (PEDOT)/poly(styrene sulfonate) (PSS) polyelectrolyte into giant unilamellar phospholipid vesicles (GUVs) and cross-linking with Ca2+ ions to generate a hydrogel within the internal compartment are reported. The aqueous colloidal suspension of PEDOT with excess PSS was microinjected into the internal compartment of liposomes as well as networks of GUVs and lipid nanotubes. The subsequent introduction of calcium ions as cross-linking agent in order to induce hydrogel formation was achieved by three different methods: vesicle fusion, electroporation, and direct microinjection. Gel formation was probed by coinjection of fluorescent nanoparticles and tracking of Brownian motion. Particle mobility was shown to be distinctly reduced in the gel-filled vesicles. Diffusion constants for the particles were calculated from the projected movement of the particles and compared to particles in reference gels and solutions.     

Water diffusion through a membrane protein channel: a first passage time approach

Water diffusion through OmpF, a porin in the outer membrane of Escherichia coli, is studied by molecular dynamics simulation. A first passage time approach allows characterizing the diffusive properties of a well-defined region of this channel. A carbon nanotube, which is considerably more homogeneous, serves as a model to validate the methodology. Here we find, in addition to the expected regular behavior, a gradient of the diffusion coefficient at the channel ends, witness of the transition from confinement in the channel to bulk behavior in the connected reservoirs. Moreover, we observe the effect of a kinetic boundary layer, which is the counterpart of the initial ballistic regime in a mean square displacement analysis. The overall diffusive behavior of water in OmpF shows remarkable similarity with that in a homogeneous channel. However, a small fraction of the water molecules appears to be trapped by the protein wall for considerable lengths of time. The distribution of trapping times exhibits a broad power law distribution psi(tau) approximately tau (-2.4), up to tau=10 ns, a bound set by the length of the simulation run. We discuss the effect of this distribution on the dynamic properties of water in OmpF in terms of incomplete sampling of phase space.     

Quantum mechanical calculations on selectivity in the KcsA channel: the role of the aqueous cavity

We have carried out quantum calculations on selected residues at the intracellular side of the selectivity filter of the KcsA potassium channel, using the published X-ray coordinates as starting points. The calculations involved primarily the side chains of residues lining the aqueous cavity on the intracellular side of the selectivity filter, in addition to water molecules, plus a K+ or Na+ ion. The results showed unambiguously that Na+ significantly distorts the symmetry of the channel at the entrance to the selectivity filter (at the residue T75), while K+ does so to a much smaller extent. In all, three ion positions have been calculated: the S4 (lowest) position at the bottom of the selectivity filter, the top of the cavity, and the midpoint of the cavity; Na+ is trapped at the cavity top, while K+ is cosolvated by the selectivity filter carbonyl groups plus threonine hydroxyl groups so that it can traverse the filter. Only one water molecule remains in the K+ solvation shell at the upper position in the cavity; this solvation shell also contains four threonine (T75) hydroxyl oxygens and two backbone carbonyls, while Na+ is solvated by five molecules of water and one oxygen from threonine hydroxyls. T75 at the entrance to the selectivity filter has a key role in recognition of the alkali ion, and T74 has secondary importance. The energetic basis for the preferential bonding of potassium by these residues is briefly discussed, based on additional calculations. Taken together, the results suggest that Na+ would have difficulty entering the cavity, and if it did, it would not be able to enter the selectivity filter.     

Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy

A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normal-mode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.     

Spontaneous vesicle self-assembly: a mesoscopic view of membrane dynamics

Amphiphilic vesicles are ubiquitous in living cells and industrially interesting as drug delivery vehicles. Vesicle self-assembly proceeds rapidly from nanometer to micrometer length scales and is too fast to image experimentally but too slow for molecular dynamics simulations. Here, we use parallel dissipative particle dynamics (DPD) to follow spontaneous vesicle self-assembly for up to 445 μs with near-molecular resolution. The mean mass and radius of gyration of growing amphiphilic clusters obey power laws with exponents of 0.85 ± 0.03 and 0.41 ± 0.02, respectively. We show that DPD provides a computational window onto fluid dynamics on scales unreachable by other explicit-solvent simulations.     

Surface planar bilayers of phospholipids used in protein membrane reconstitution: an atomic force microscopy study

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.     

Prediction of axial concentration profiles in an extractive membrane bioreactor and experimental verification

A steady-state mathematical model has been developed to predict axial-concentration profiles of a pollutant in an extractive-membrane bioreactor (EMB). Typically, the previous models describe the pollutant concentration profiles in the membrane-attached biofilms in a direction perpendicular to the membrane. In contrast, the model presented in this work describes not only the radial profiles, but also the axial profiles along the membrane length. Biofilms of Xanthobacter autotrophicus GJ10 were grown on the surface of silicone rubber tubes. A diffusion-reaction model was employed to describe the diffusion and reaction in the biofilm in the radial direction. Membrane tubes were modelled as a series of mixed tanks to allow the prediction of axial concentrations. The model predictions were verified by experimental data from a range of operating conditions. These included different dissolved oxygen concentrations in biomedium and different wastewater flowrates. Finally, the rate-limiting step in the reactor was determined to be the mass-transfer resistance of the pollutant in the biofilm.     

Vesicle fission of giant unilamellar vesicles of liquid-ordered-phase membranes induced by amphiphiles with a single long hydrocarbon chain

Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single giant unilamellar vesicle (GUV) method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine/cholesterol(6/4) (DPPC/chol(6/4)) membranes and sphingomyelin/cholesterol membranes (6/4) in the liquid-ordered (lo) phase. In this report, to elucidate its mechanism, we have investigated the effect of low concentrations (much less than their CMC) of other amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) on DPPC/chol(6/4) GUVs as well as the effect of the membrane composition on the lyso-PC-induced vesicle fission. We found that low concentrations of single long chain amphiphiles (lyosophosphatidic acid, octylglucoside, and sodium dodecyl sulfate) induced the shape change from a prolate to two spheres connected by a very narrow neck, indicating that the single long chain amphiphiles can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. As the single long chain amphiphile concentrations were increased, all of them induced vesicle fission of DPPC/chol(6/4) GUVs above their threshold concentrations. To elucidate the role of cholesterol in the single long chain amphiphile-induced vesicle fission, we investigated the effect of lyso-PC on GUVs of dioleoyl-PC (DOPC)/chol(6/4) membranes in the Lalpha phase; no vesicle fission occurred, indicating that cholesterol in itself did not play an important role in the vesicle fission. Finally, to elucidate the effect of the inclusion of DOPC in the lo-phase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC increased. At and above 30 mol % DOPC, no vesicle fission occurred. On the basis of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of a GUV of a lo-phase membrane.     

Bead-linked proteoliposomes: a reconstitution method for nmr analyses of membrane protein-ligand interactions

Structural information about the interactions between membrane proteins and their ligands provides insights into the membrane protein functions. A variety of surfactants have been used for structural analyses of membrane proteins, and in some cases, they yielded successful results. However, the use of surfactants frequently increases the conformational instability of membrane proteins and distorts their normal function. Here, we propose a new strategy of membrane protein reconstitution into lipid bilayers on affinity beads, which maintains the native conformation and function of the protein for NMR studies. The reconstituted membrane proteins are suitable for NMR analyses of interactions, by using the transferred cross-saturation method. The strategy was successfully applied to the interaction between a potassium ion channel, KcsA, and a pore-blocker, agitoxin2 (AgTx2). This strategy would be useful for analyzing the interactions between various membrane proteins and their ligands.     

Incorporation of the HERG potassium channel in a mercury supported lipid bilayer

The HERG potassium channel was incorporated in a mercury-supported tethered bilayer lipid membrane (tBLM) obtained by anchoring a thiolipid monolayer to the mercury surface and by self-assembling a lipid monolayer on top of it from a lipid film spread on the surface of an electrolyte solution. HERG was then incorporated in this tBLM from its micellar solution in Triton X-100, thus avoiding the use of vesicles in the preparation of the tBLM and of proteoliposomes in channel incorporation. The HERG "inward" current following a repolarization step was obtained by subtracting the current recorded upon addition of the specific inhibitor WAY from that recorded prior to this addition. This current was compared with that reported in the literature by the patch-clamp technique.     

Insight into the origins of the barrier-less knock-on conduction in the KcsA channel: molecular dynamics simulations and ab initio calculations

Since the pioneering work of Zhou et al. (Y. Zhou, J. H. Morais-Cabral, A. Kaufman and R. MacKinnon, Nature, 2001, 414, 43-48) it is now well established that the streptomyces lividans potassium channel (KcsA) can accommodate more than one ion, namely between 2 and 3. As a result, it is usually assumed that the conduction of ions proceeds through a barrier-less knock-on mechanism. This one is an alternation of two sequences containing either 2 or 3 ions which have nearly the same energies. However, the origin of such knock-on mechanism is not clearly known. The entry and the exit of ion in or out of the selectivity filter are suspected to be due to the repulsive interactions between ions. In this work, molecular dynamics simulations running over nanoseconds have been done in order to identify such events. Two specific situations, namely (S(1), S(3)) containing 2 ions and (S(2), S(4)) containing 3 ions, have been investigated regarding the different locations that ions can occupy during their diffusion through the selectivity filter of KcsA. We show that contractions of the (S(1), S(3)) file and dilation of the (S(2), S(4)) file are at the origin of the passage from one sequence to the other. The comparison between the experimentally observed diffusion rate and the occurrence's frequency of such contractions or dilation confirm the importance of such events. Ab initio calculations have also been conducted in order to examine the effect of ion polarization in the filter of KcsA. During the contraction of the ion/water file, one charge at the extra-cellular mouth of the channel strongly deviates from the others. This behavior could guide the diffusion direction to a certain extent since the contraction of the (S(1), S(3)) is favored.