Thermodynamic measurements of bilayer insertion of a single transmembrane helix chaperoned by fluorinated surfactants

Accurate determination of the free energy of transfer of a helical segment from an aqueous into a transmembrane (TM) conformation is essential for understanding and predicting the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic TM peptides were impossible due to peptide aggregation outside the lipid bilayer. Here, we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation but, unlike detergents, do not themselves interact with the bilayer. We have applied the fluorescence correlation spectroscopy methodology to study surfactant-chaperoned insertion into preformed POPC (palmitoyloleoylphosphatidylcholine) vesicles of the two well-studied dye-labeled TM peptides of different lengths: WALP23 and WALP27. Extrapolation of the apparent free-energy values measured in the presence of surfactants to a zero surfactant concentration yielded free-energy values of -9.0±0.1 and -10.0±0.1 kcal/mol for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed helical structure of peptides in lipid bilayer, in the presence of surfactants, and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements, we conclude that the partitioning of a four-residue L-A-L-A segment in the context of a continuous helical conformation from an aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal/mol. Our measurements, combined with the predictions of two independent experimental hydrophobicity scales, indicate that the per-residue cost of transfer of the helical backbone from water to the hydrocarbon core of the lipid bilayer is unfavorable and is equal to +2.13±0.17 kcal/mol.     

Theoretical calculations of the permeability of monensin-cation complexes in model bio-membranes

Monensin is one of the best-characterized ionophores; it functions in the electroneutral exchange of cations between the extracellular and cytoplasmic sides of cell membranes. The X-ray crystal structures of monensin in free acid form and in complex with Na(+), K(+) and Ag(+) are known and we have recently measured the diffusion rates of monensin in free acid form (Mo-H) and in complex with Na(+) (Mo-Na) and with K(+) (Mo-K) using laser pulse techniques. The results have shown that Mo-H diffuses across the membrane one order of magnitude faster than Mo-Na and two orders of magnitude faster than Mo-K. Here, we report calculations of the translocation free energy of these complexes across the membrane along the most favorable path, i.e. the lowest free energy path. The calculations show that the most favorable orientation of monensin is with its hydrophobic furanyl and pyranyl moieties in the hydrocarbon region of the membrane and the carboxyl group and the cation at the water-membrane interface. Further, the calculations show that Mo-H is likely to be inserted deeper than Mo-Na into the bilayer, and that the free energy barrier for transfer of Mo-H across the membrane is approximately 1 kcal/mol lower than for Mo-Na, in good agreement with our measurements. Our results show that the Mo-K complex is unlikely to diffuse across lipid bilayers in its X-ray crystal structure, in contrast to the Mo-H and Mo-Na complexes. Apparently, when diffusing across the membrane, the Mo-K complex assumes a different conformation and/or thinning defects in the bilayer lower significantly the free energy barrier for the process. The suitability of the model for treating the membrane association of small molecules is discussed in view of the successes and failures observed for the monensin system.     

Interactions of cholesterol with lipid bilayers: the preferred configuration and fluctuations.

The free energy difference associated with the transfer of a single cholesterol molecule from the aqueous phase into a lipid bilayer depends on its final location, namely on its insertion depth and orientation within the bilayer. We calculated desolvation and lipid bilayer perturbation contributions to the water-to-membrane transfer free energy, thus allowing us to determine the most favorable location of cholesterol in the membrane and the extent of fluctuations around it. The electrostatic and nonpolar contributions to the solvation free energy were calculated using continuum solvent models. Lipid layer perturbations, resulting from both conformational restrictions of the lipid chains in the vicinity of the (rigid) cholesterol backbone and from cholesterol-induced elastic deformations, were calculated using a simple director model and elasticity theory, respectively. As expected from the amphipathic nature of cholesterol and in agreement with the available experimental data, our results show that at the energetically favorable state, cholesterol's hydrophobic core is buried within the hydrocarbon region of the bilayer. At this state, cholesterol spans approximately one leaflet of the membrane, with its OH group protruding into the polar (headgroup) region of the bilayer, thus avoiding an electrostatic desolvation penalty. We found that the transfer of cholesterol into a membrane is mainly driven by the favorable nonpolar contributions to the solvation free energy, whereas only a small opposing contribution is caused by conformational restrictions of the lipid chains. Our calculations also predict a strong tendency of the lipid layer to elastically respond to (thermally excited) vertical fluctuations of cholesterol so as to fully match the hydrophobic height of the solute. However, orientational fluctuations of cholesterol were found to be accompanied by both an elastic adjustment of the surrounding lipids and by a partial exposure of the hydrophobic cholesterol backbone to the polar (headgroup) environment. Our calculations of the molecular order parameter, which reflects the extent of orientational fluctuations of cholesterol in the membrane, are in good agreement with available experimental data.     

Nonclassical hydrophobic effect in membrane binding equilibria.

The enthalpy of transfer of four different amphiphilic molecules from the aqueous phase to the lipid membrane was determined by titration calorimetry. The four molecules investigated were the potential-sensitive dye 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), the membrane conductivity inducing anion tetraphenylborate (TPB), the Ca2+ channel blocker amlodipine [B?uerle, H. D., & Seelig, J. (1991) Biochemistry 30, 7203-7211], and the positively charged local anesthetic dibucaine. All four amphiphiles penetrate into the hydrophobic part of the membrane, and their binding constants, after correcting for electrostatic effects, range between 600 M-1 for dibucaine and 60,000 M-1 for tetraphenylborate. The corresponding changes in free energy were about -6 to -9 kcal/mol. Binding of the amphiphiles to membrane vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was accompanied by exothermic heats of reaction for all four molecules. For TNS, TPB, and amlodipine, the enthalpies of transfer were almost identical and corresponded to delta H approximately -9 kcal/mol, essentially accounting for the total free energy change. Thus, the binding of these charged amphiphiles to the hydrophobic membrane was driven by enthalpy. This is in contrast to the classical hydrophobic effect, where the transfer is considered to be entropy driven. For dibucaine, the enthalpy of transfer was smaller with delta H approximately -2 kcal/mol but was still about one-third of the total free energy change. All enthalpies of transfer exhibited a distinct temperature dependence with molar heat capacities delta Cp of -30 to -100 cal mol-1K-1 for the transfer from water to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Implicit solvent model estimates of the stability of model structures of the alamethicin channel

Alamethicin is a hydrophobic helical peptide of 20 residues, which oligomerizes to form ion-conducting channels in membranes. The behavior of an intact alamethicin channel in POPC bilayers was recently studied, using 2 ns molecular dynamics (MD) simulations of a model hexameric channel. These simulations produced numerous conformations of the channel. In the present study, we used 11 of these channel conformations and carried out continuum-solvent model calculations, similar to those used for the monomers in our previous studies, to investigate the energetics of the channel inside the lipid bilayer. Our results suggest that, out of the 11 channel conformations produced by the MD simulations, only four are stable inside the lipid bilayer, with water-to-membrane free energies of transfer ranging from ~–6 to ~–10 kcal/mol. Analysis of the results suggests two causes for the apparent instability of the remainder of the structures inside the lipid bilayer, both resulting from the desolvation of channel polar groups (i.e. their transfer from the aqueous phase into the bilayer). The first is specific, uncompensated backbone hydrogen bonds, which exist in the region of the channel exposed to the hydrocarbon of the lipid bilayer. The second is exposure of intra-pore water molecules to the surrounding lipid. Thus, the association of these structures with the membrane involves a large electrostatic desolvation free-energy penalty. The apparent conflict between continuum-solvent and MD calculations, and its significance for the interpretation of membrane proteins simulations, are discussed.     

Designing transmembrane alpha-helices that insert spontaneously

Direct measurement of the free energies of transfer of hydrophobic membrane-spanning alpha-helices from water to membranes is important for the determination of an accurate experiment-based hydrophobicity scale for membrane proteins. An important objective of such a scale is to account for the presently unknown thermodynamic cost of partitioning hydrogen-bonded peptide bonds into the membrane hydrocarbon core. We describe here the physical properties of a transmembrane (TM) peptide, TMX-1, designed to test the feasibility of engineering peptides that spontaneously insert across bilayers but that have the important property of measurable monomeric water solubility. TMX-1, Ac-WNALAAVAAAL-AAVAAALAAVAAGKSKSKS-NH(2), is a 31-residue sequence with a 21-residue nonpolar core, N- and C-caps to favor helix formation, and a highly polar C-terminus to improve solubility and to control directionality of insertion into lipid vesicles. TMX-1 appeared to be soluble in water up to a concentration of at least 1 mg/mL (0.3 mM). However, fluorescence spectroscopy, fluorescence quenching, and circular dichroism (CD) spectroscopy indicated that the high solubility was due to the formation of molecular aggregates that persisted at peptide concentrations down to at least 0.1 microM peptide. Nevertheless, aqueous TMX-1 partitioned strongly into membrane vesicles with apparent mole-fraction free-energy values of -7.1 kcal mol(-1) for phosphatidylcholine (POPC) vesicles and -8.2 kcal mol(-1) for phosphatidylglycerol (POPG) vesicles. CD spectroscopy of TMX-1 in oriented multilayers formed from either lipid disclosed a very strong preference for a transmembrane alpha-helical conformation. When TMX-1 was added to preformed vesicles, it was fully helical. A novel fluorescence resonance energy transfer (FRET) method demonstrated that at least 50% of the TMX-1 insered spontaneously across the vesicle membranes. Binding and insertion were found to be fully reversible for POPC vesicles but not POPG vesicles. TMX-1 was thus found to have many of the properties required for thermodynamic measurements of TM peptide insertion. Importantly, the results obtained delineate the experimental problems that must be considered in the design of peptides that can partition spontaneously and reversibly as monomers into and across membranes. Our success with TMX-1 suggests that these problems are not insurmountable.     

Membrane composition modulates thiopental partitioning in bilayers and biomembranes

The nonspecific interaction of thiopental with erythrocyte ghosts, synaptic membranes, microsomes and mitochondria has been measured at 25°C and pH 6.6. In cholesterol-depleted erythrocyte ghosts the partition coefficient decreases with increasing cholesterol content. In sonicated liposomes made from egg lecithin and cholesterol the partition coefficient also decreases with increasing cholesterol content. The dependence of the partition coefficient on cholesterol content in the biological membranes, on average, parallels that in the lipid bilayers. The partition coefficient in lipid bilayers made from lipids extracted from erythrocyte ghosts was comparable to that in the corresponding egg lecithin/cholesterol bilayer. The partition coefficients of all the biomembranes are consistently lower than those in the corresponding egg lecithin/cholesterol bilayer, the free energy of transfer between biomembrane and corresponding bilayer being ?1 kcal/mol.     

Probing red cell membrane cholesterol movement with cyclodextrin

We probed the kinetics with which cholesterol moves across the human red cell bilayer and exits the membrane using methyl-beta-cyclodextrin as an acceptor. The fractional rate of cholesterol transfer (% s(-1)) was unprecedented, the half-time at 37 degrees C being ~1 s. The kinetics observed under typical conditions were independent of donor concentration and directly proportional to acceptor concentration. The rate of exit of membrane cholesterol fell hyperbolically to zero with increasing dilution. The energy of activation for cholesterol transfer was the same at high and low dilution; namely, 27-28 Kcal/mol. This behavior is not consistent with an exit pathway involving desorption followed by aqueous diffusion to acceptors nor with a simple one-step collision mechanism. Rather, it is that predicted for an activation-collision mechanism in which the reversible partial projection of cholesterol molecules out of the bilayer precedes their collisional capture by cyclodextrin. Because the entire membrane pool was transferred in a single first-order process under all conditions, we infer that the transbilayer diffusion (flip-flop) of cholesterol must have proceeded faster than its exit, i.e., with a half-time of <1 s at 37 degrees C.     

Interaction of octyl-beta-thioglucopyranoside with lipid membranes.

Octyl-beta-thioglucopyranoside (octyl thioglucoside, OTG) is a nonionic surfactant used for the purification, reconstitution, and crystallization of membrane proteins. The thermodynamic properties of the OTG-membrane partition equilibrium are not known and have been investigated here with high-sensitivity titration calorimetry. The critical concentration for inducing the bilayer <==> micelle transition was determined as cD* = 7.3 mM by 90 degree light scattering. All thermodynamic studies were performed well below this limit. Sonified, unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol were employed in the titration calorimetry experiments, and the temperature was varied between 28 degrees C and 45 degrees C. Depending on the surfactant concentration in the membrane, the partition enthalpy was found to be exothermic or endothermic, leading to unusual titration patterns. A quantitative interpretation of all titration curves was possible with the following model: 1) The partitioning of OTG into the membrane follows a simple partition law, i.e., Xb = Kc(D,f), where Xb denotes the molar amount of detergent bound per mole of lipid and c(D,f) is the detergent concentration in bulk solution. 2) The partition enthalpy for the transfer of OTG from the aqueous phase to the membrane depends linearly on the mole fraction, R, of detergent in the membrane. All calorimetric OTG titration curves can be characterized quantitatively by using a composition-dependent partition enthalpy of the form deltaHD(R) = -0.08 + 1.7 R (kcal/mol) (at 28 degrees C). At low OTG concentrations (R < or = 0.05) the reaction enthalpy is exothermic; it becomes distinctly endothermic as more and more surfactant is incorporated into the membrane. OTG has a partition constant of 240 M(-1) and is more hydrophobic than its oxygen-containing analog, octyl-beta-D-glucopyranoside (OG). Including a third nonionic amphiphile, octa(ethyleneoxide) dodecylether (C12EO8), an empirical relation can be established between the Gibbs energies of membrane partitioning, deltaGp, and micelle formation, deltaGmic, with deltaGp = 1.398 + 0.647 deltaGmic (kcal/mol). The partition constant of OTG is practically independent of temperature and of the cholesterol content of the membrane. In contrast, the partition enthalpy shows a strong temperature dependence. The molar specific heat capacity of the transfer of OTG from the aqueous phase to the membrane is deltaCp = -98 cal/(mol x K). The OTG partition enthalpy is also dependent on the cholesterol content of the membrane. It increases by approximately 1 kcal/mol at 50 mol% cholesterol. As the partition constant remains unchanged, the increase in enthalpy is compensated for by a corresponding increase in entropy, presumably caused by a restructuring of the membrane hydration layer.     

Interaction and structural study of kinin peptide bradykinin and ganglioside monosialylated 1 micelle

Partitioning of small proteins and peptides from the aqueous to membrane phase is often coupled with folding. In this work we examine the binding and folding of the kinin peptide, bradykinin (BK), in the presence of the ganglioside monosialylated 1 (GM1) micelle. Using two-dimensional NMR techniques, we have shown that at low concentration, GM1 micelle is able to induce a turn conformation to BK. A pulsed-field gradient diffusion NMR study indicated that the peptide partitions into the GM1 micelle with a DeltaG(part) of -3.14 +/- 0.03 kcal/mol. A saturation transfer difference (STD) NMR study indicated that the binding is mostly through hydrophobic residues.     

Amphipathic helixes and plasma lipoproteins: Thermodynamic and geometric considerations

In this paper analyses are made of the thermodynamic and geometric properties of the predicted association between amphipathic helixes and phospholipid vesicles. From thermodynamic considerations it is proposed that a major driving force for such an association is the negative free energy gained by the transfer of a number of hydrophobic residues (contained within the non-polar faces of amphipathic helixes), from water to the interior of a phospholipid bilayer. The mechanism proposed is that in the aqueous state a potentially amphipathic sequence forms a non-helical hydrophobic patch on the surface of the apolipoprotein. Formation of an amphipathic helix and simultaneous burial of the hydrophobic residues in the surface of a phospholipid bilayer provides the driving force for lipid association. From this model an estimate of the upperlimit for the hydrophobically driven free energy of lipid association (?40?65 kcal/mol) is calculated for the 4 apolipoproteins with known sequences.On the basis of geometrical considerations a model for an intermediate state of high density lipoprotein (HDL) synthesis is proposed. This model consists of a cholesterol-containing phospholipid bilayer disc whose ‘naked’ hydrophobic edges are shielded from the aqueous phase by amphipathic helixes of the apolipoproteins. Exposure of these ‘bicycle tire’ micelles to the enzyme lecithin: cholesterol acyl transferase (LCAT) is postulated to result in the formation of mature spherical HDL particles with cholesteryl ester forming a neutral lipid core.     

Theoretical calculation of the binding free energies for pyruvate dehydrogenase E1 binding with ligands

We have tested a computational protocol based on molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free-energy calculations to examine the detailed microscopic structures and binding free energies for the pyruvate dehydrogenase multienzyme complex (PDHc) E1 binding with its ligands (cofactor and inhibitors). The calculated binding free energies are all in good agreement with available experimental data, with an average absolute deviation of approximately 0.7 kcal/mol, suggesting that the computational protocol tested may be valuable in future rational design of new, more potent inhibitors of PDHc E1.     

Continuum solvent model calculations of alamethicin-membrane interactions: thermodynamic aspects

Alamethicin is a 20-amino acid antibiotic peptide that forms voltage-gated ion channels in lipid bilayers. Here we report calculations of its association free energy with membranes. The calculations take into account the various free-energy terms that contribute to the transfer of the peptide from the aqueous phase into bilayers of different widths. The electrostatic and nonpolar contributions to the solvation free energy are calculated using continuum solvent models. The contributions from the lipid perturbation and membrane deformation effects and the entropy loss associated with peptide immobilization in the bilayer are estimated from a statistical thermodynamic model. The calculations were carried out using two classes of experimentally observed conformations, both of which are helical: the NMR and the x-ray crystal structures. Our calculations show that alamethicin is unlikely to partition into bilayers in any of the NMR conformations because they have uncompensated backbone hydrogen bonds and their association with the membrane involves a large electrostatic solvation free energy penalty. In contrast, the x-ray conformations provide enough backbone hydrogen bonds for the peptide to associate with bilayers. We tested numerous transmembrane and surface orientations of the peptide in bilayers, and our calculations indicate that the most favorable orientation is transmembrane, where the peptide protrudes approximately 4 A into the water-membrane interface, in very good agreement with electron paramagnetic resonance and oriented circular dichroism measurements. The calculations were carried out using two alamethicin isoforms: one with glutamine and the other with glutamate in the 18th position. The calculations indicate that the two isoforms have similar membrane orientations and that their insertion into the membrane is likely to involve a 2-A deformation of the bilayer, again, in good agreement with experimental data. The implications of the results for the biological function of alamethicin and its capacity to oligomerize and form ion channels are discussed.     

Free-energy profiles of membrane insertion of the M2 transmembrane peptide from influenza A virus

The insertion of the M2 transmembrane peptide from influenza A virus into a membrane has been studied with molecular-dynamics simulations. This system is modeled by an atomically detailed peptide interacting with a continuum representation of a membrane bilayer in aqueous solution. We performed replica-exchange molecular-dynamics simulations with umbrella-sampling techniques to characterize the probability distribution and conformation preference of the peptide in the solution, at the membrane interface, and in the membrane. The minimum in the calculated free-energy surface of peptide insertion corresponds to a fully inserted, helical peptide spanning the membrane. The free-energy profile also shows that there is a significant barrier for the peptide to enter into this minimum in a nonhelical conformation. The sequence of the peptide is such that hydrophilic amino acid residues at the ends of the otherwise primarily hydrophobic peptide create a trapped, U-shaped conformation with the hydrophilic residues associated with the aqueous phase and the hydrophobic residues embedded in the membrane. Analysis of the free energy shows that the barrier to insertion is largely enthalpic in nature, whereas the membrane-spanning global minimum is favored by entropy.     

Partitioning of 2,6-Bis(1H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.     

Study on the binding mode of a pyrrolotriazin derivative with JAK2 by docking and MD simulation

The pyrrolotriazin derivative 2-(4-(4-((7-(3-(N-methylmethylsulfonamido)phenyl)pyrrolo [2,1-f][1,2,4]triazin-2-yl)amino)phenyl)piperidin-1-yl)acetamide (PPA) is a potential Janus kinase 2 (JAK2) inhibitor. The binding mode between PPA and JAK2 was investigated by using a combined method of docking, molecular dynamics (MD) simulation and binding free-energy calculation. The docking calculations preliminarily indicated that there were two possible binding modes 1 and 2; MD simulations and binding free-energy calculations identified that binding mode 1 was more stable and favourable, with the lower MM-PBSA binding free energy of ?34.00?±?0.17?kcal/mol. Moreover, some valuable binding information is revealed as follows: the inhibitor PPA is suitably located at the ATP-binding site of JAK2 and the hydrophobic interaction plays an essential role. PPA not only interacts with residues Leu855, Val863, Ala880, Tyr931, Leu932 and Leu983 via hydrophobic interaction but also interacts with Ser936 and Asp994 by hydrogen bonds. These two factors are advantageous for PPA to strongly bind to JAK2. These results help to understand the action mechanisms and designing new compounds with a higher affinity to JAK2.     

Mechanism for the transport of ammonia within carbamoyl phosphate synthetase determined by molecular dynamics simulations

Carbamoyl phosphate synthetase (CPS) is a member of the amidotransferase family of enzymes that uses the hydrolysis of glutamine as a localized source of ammonia for biosynthetic transformations. Molecular dynamics simulations for the transfer of ammonia and ammonium through a tunnel in the small subunit of CPS resulted in five successful trajectories for ammonia transfer, while ammonium was immobilized in a water pocket inside the small subunit of the heterodimeric protein. The observed molecular tunnel for ammonia transport is consistent with that suggested by earlier X-ray crystallography and site-directed mutation studies. His-353, Ser-47, and Lys-202, around the active site center in the small subunit, function cooperatively to deliver ammonia from the site of formation to the interface with the large subunit, via the exchange of hydrogen bonds with a critical water cluster within the tunnel. The NH 3 forms and breaks hydrogen bonds to Gly-292, Ser-35, Pro-358, Gly-293, and Thr-37 in a stepwise fashion "macroscopically" as it travels through the hydrophilic passage toward the subunit interface. The potential of mean force calculations along the ammonia transfer pathway indicates a low free-energy path for the translocation of ammonia with two barriers of 3.9 and 5.5 kcal/mol, respectively. These low free-energy barriers are consistent with the delivery of ammonia from the site of formation into a water reservoir toward the exit of the tunnel and migration through the hydrophilic leaving passage, respectively. The high overall free-energy barrier of 22.4 kcal/mol for the transport of ammonium additionally substantiates that the tunnel in the small subunit of CPS is not an ammonium but an ammonia channel.     

Free energy of membrane protein unfolding derived from single-molecule force measurements

Mechanical single-molecule techniques offer exciting possibilities to investigate protein folding and stability in native environments at submolecular resolution. By applying a free-energy reconstruction procedure developed by Hummer and Szabo, which is based on a statistical theorem introduced by Jarzynski, we determined the unfolding free energy of the membrane proteins bacteriorhodopsin (BR), halorhodopsin, and the sodium-proton antiporter NhaA. The calculated energies ranged from 290.5 kcal/mol for BR to 485.5 kcal/mol for NhaA. For the remarkably stable BR, the equilibrium unfolding free energy was independent of pulling rate and temperature ranging between 18 and 42 degrees C. Our experiments also revealed heterogeneous energetic properties in individual transmembrane helices. In halorhodopsin, the stabilization of a short helical segment yielded a characteristic signature in the energy profile. In NhaA, a pronounced peak was observed at a functionally important site in the protein. Since a large variety of single- and multispan membrane proteins can be tackled in mechanical unfolding experiments, our approach provides a basis for systematically elucidating energetic properties of membrane proteins with the resolution of individual secondary-structure elements.     

Stability of an ion channel in lipid bilayers: implicit solvent model calculations with gramicidin

Gramicidin is a helical peptide, 15 residues in length, which dimerizes to form ion-conducting channels in lipid bilayers. Here we report calculations of its free energy of transfer from the aqueous phase into bilayers of different widths. The electrostatic and nonpolar contributions to the desolvation free energy were calculated using implicit solvent models, in which gramicidin was described in atomic detail and the hydrocarbon region of the membrane was described as a slab of hydrophobic medium embedded in water. The free energy penalties from the lipid perturbation and membrane deformation effects, and the entropy loss associated with gramicidin immobilization in the bilayer, were estimated from a statistical thermodynamic model of the bilayer. The calculations were carried out using two classes of experimentally observed conformations: a head-to-head dimer of two single-stranded (SS) beta-helices and a double-stranded (DS) intertwined double helix. The calculations showed that gramicidin is likely to partition into the bilayer in all of these conformations. However, the SS conformation was found to be significantly more stable than the DS in the bilayer, in agreement with most of the experimental data. We tested numerous transmembrane and surface orientations of gramicidin in bilayers of various widths. Our calculations indicate that the most favorable orientation is transmembrane, which is indeed to be expected from a channel-forming peptide. The calculations demonstrate that gramicidin insertion into the membrane is likely to involve a significant deformation of the bilayer to match the hydrophobic width of the peptide (22 A), again in good agreement with experimental data. Interestingly, deformation of the bilayer was induced by all of the gramicidin conformations.