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Olaf S Andersen

Publications and source records attributed to Olaf S Andersen.

15 recordsLinked to original sources
PubMed2006

2,3-butanedione monoxime affects cystic fibrosis transmembrane conductance regulator channel function through phosphorylation-dependent and phosphorylation-independent mechanisms: the role of bilayer material properties.

2,3-Butanedione monoxime (BDM) is widely believed to act as a chemical phosphatase. We therefore examined the effects of BDM on the cystic fibrosis transmembrane regulator (CFTR) Cl(-) channel, which is regulated by phosphorylation in a complex manner. In guinea pig ventricular myocytes, forskolin-activated whole-cell CFTR currents responded biphasically to external 20 mM BDM: a rapid approximately 2-fold current activation was followed by a slower (tau approximately 20 s) inhibition (to approximately 20% of control). The inhibitory response was abolished by intracellular dialysis with the phosphatase inhibitor microcystin, suggesting involvement of endogenous phosphatases. The BDM-induced activation was studied further in Xenopus laevis oocytes expressing human epithelial CFTR. The concentration for half-maximal BDM activation (K(0.5)) was state-dependent, approximately 2 mM for highly and approximately 20 mM for partially phosphorylated channels, suggesting a modulated receptor mechanism. Because BDM modulates many different membrane proteins with similar K(0.5) values, we tested whether BDM could alter protein function by altering lipid bilayer properties rather than by direct BDM-protein interactions. Using gramicidin channels of different lengths (different channel-bilayer hydrophobic mismatch) as reporters of bilayer stiffness, we found that BDM increases channel appearance rates and lifetimes (reduces bilayer stiffness). At 20 mM BDM, the appearance rates increase approximately 4-fold (for the longer, 15 residues/monomer, channels) to approximately 10-fold (for the shorter, 13 residues/monomer channels); the lifetimes increase approximately 50% independently of channel length. BDM thus reduces the energetic cost of bilayer deformation, an effect that may underlie the effects of BDM on CFTR and other membrane proteins; the state-dependent changes in K(0.5) are consistent with such a bilayer-mediated mechanism.

Animals↗
PubMed2006

Molecular dynamics - potential of mean force calculations as a tool for understanding ion permeation and selectivity in narrow channels.

Ion channels catalyze the permeation of charged molecules across cell membranes and are essential for many vital physiological functions, including nerve and muscle activity. To understand better the mechanisms underlying ion conduction and valence selectivity of narrow ion channels, we have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the prototypical gramicidin A channel. Employing modern all-atom molecular dynamics (MD) force fields with umbrella sampling methods that incorporate one hundred 1-2 ns trajectories, we find that it is possible to achieve semi-quantitative agreement with experimental binding and conductance measurements. We also examine the sensitivity of the MD-PMF results to the choice of MD force field and compare PMFs for potassium, calcium and chloride ions to explore the basis for the valence selectivity of this narrow and uncharged ion channel. A large central barrier is observed for both anions and divalent ions, consistent with lack of experimental conductance. Neither anion or divalent cation is seen to be stabilized inside the channel relative to the bulk electrolyte and each leads to large disruptions to the protein and membrane structure when held deep inside the channel. Weak binding of calcium ions outside the channel corresponds to a free energy well that is too shallow to demonstrate channel blocking. Our findings emphasize the success of the MD-PMF approach and the sensitivity of ion energetics to the choice of biomolecular force field.

Barium↗
PubMed2006

Ion permeation through a narrow channel: using gramicidin to ascertain all-atom molecular dynamics potential of mean force methodology and biomolecular force fields.

We investigate methods for extracting the potential of mean force (PMF) governing ion permeation from molecular dynamics simulations (MD) using gramicidin A as a prototypical narrow ion channel. It is possible to obtain well-converged meaningful PMFs using all-atom MD, which predict experimental observables within order-of-magnitude agreement with experimental results. This was possible by careful attention to issues of statistical convergence of the PMF, finite size effects, and lipid hydrocarbon chain polarizability. When comparing the modern all-atom force fields of CHARMM27 and AMBER94, we found that a fairly consistent picture emerges, and that both AMBER94 and CHARMM27 predict observables that are in semiquantitative agreement with both the experimental conductance and dissociation coefficient. Even small changes in the force field, however, result in significant changes in permeation energetics. Furthermore, the full two-dimensional free-energy surface describing permeation reveals the location and magnitude of the central barrier and the location of two binding sites for K(+) ion permeation near the channel entrance--i.e., an inner site on-axis and an outer site off-axis. We conclude that the MD-PMF approach is a powerful tool for understanding and predicting the function of narrow ion channels in a manner that is consistent with the atomic and thermally fluctuating nature of proteins.

Cell Membrane Permeability↗
PubMed2005

Gramicidin channels.

Gramicidin channels are mini-proteins composed of two tryptophan-rich subunits. The conducting channels are formed by the transbilayer dimerization of nonconducting subunits, which are tied to the bilayer/solution interface through hydrogen bonds between the indole NH groups and the phospholipid backbone and water. The channel structure is known at atomic resolution and the channel's permeability characteristics are particularly well defined: gramicidin channels are selective for monovalent cations, with no measurable permeability to anions or polyvalent cations; ions and water move through a pore whose wall is formed by the peptide backbone; and the single-channel conductance and cation selectivity vary when the amino acid sequence is varied, even though the permeating ions make no contact with the amino acid side chains. Given the amount of experimental information that is available--for both the wild-type channels and for channels formed by amino acid-substituted gramicidin analogues--gramicidin channels provide important insights into the microphysics of ion permeation through bilayer-spanning channels. For the same reason, gramicidin channels constitute the system of choice for evaluating computational strategies for obtaining mechanistic insights into ion permeation through the complex channels formed by integral membrane proteins.

Animals↗
PubMed2004

Bilayer-dependent inhibition of mechanosensitive channels by neuroactive peptide enantiomers.

The peptide GsMTx4, isolated from the venom of the tarantula Grammostola spatulata, is a selective inhibitor of stretch-activated cation channels (SACs). The mechanism of inhibition remains unknown; but both GsMTx4 and its enantiomer, enGsMTx4, modify the gating of SACs, thus violating a trademark of the traditional lock-and-key model of ligand-protein interactions. Suspecting a bilayer-dependent mechanism, we examined the effect of GsMTx4 and enGsMTx4 on gramicidin A (gA) channel gating. Both peptides are active, and the effect increases with the degree of hydrophobic mismatch between bilayer thickness and channel length, meaning that GsMTx4 decreases the energy required to deform the boundary lipids adjacent to the channel. GsMTx4 decreases inward SAC single-channel currents but has no effect on outward currents, suggesting it is located within a Debye length of the outer vestibule of the SACs, but significantly farther from the inner vestibule. Likewise, GsMTx4 decreases gA single-channel currents. Our results suggest that modulation of membrane proteins by amphipathic peptides--mechanopharmacology--involves not only the protein itself but also the surrounding lipids. The surprising efficacy of the d form of GsMTx4 peptide has important therapeutic implications, because d peptides are not hydrolysed by endogenous proteases and may be administered orally.

Amino Acid Sequence↗
PubMed2004

Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol.

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a "conformational" change (the monomer<-->dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.

Adaptation, Physiological↗
PubMed2003

Energetics of ion conduction through the gramicidin channel.

The free energy governing K(+) conduction through gramicidin A channels is characterized by using over 0.1 micros of all-atom molecular dynamics simulations with explicit solvent and membrane. The results provide encouraging agreement with experiments and insights into the permeation mechanism. The free energy surface of K(+), as a function of both axial and radial coordinates, is calculated. Correcting for simulation artifacts due to periodicity and the lack of hydrocarbon polarizability, the calculated single-channel conductance for K(+) ions is 0.8 pS, closer to experiment than any previous calculation. In addition, the estimated single ion dissociation constants are within the range of experimental determinations. The relatively small free energy barrier to ion translocation arises from a balance of large opposing contributions from protein, single-file water, bulk electrolyte, and membrane. Mean force decomposition reveals a remarkable ability of the single-file water molecules to stabilize K(+) by -40 kcal/mol, roughly half the bulk solvation free energy. The importance of the single-file water confirms the conjecture of Mackay et al. [Mackay, D. H. J., Berens, P. H., Wilson, K. R. & Hagler, A. T. (1984) Biophys. J. 46, 229-248]. Ion association with the channel involves gradual dehydration from approximately six to seven water molecules in the first shell, to just two inside the narrow pore. Ion permeation is influenced by the orientation of the single-file water column, which can present a barrier to conduction and give rise to long-range coupling of ions on either side of the pore. Small changes in the potential function, including contributions from electronic polarization, are likely to be sufficient to obtain quantitative agreement with experiments.

Amino Acid Sequence↗
PubMed2003

Genistein can modulate channel function by a phosphorylation-independent mechanism: importance of hydrophobic mismatch and bilayer mechanics.

Genistein, a generic tyrosine kinase inhibitor, has been used extensively as a tool to investigate the possible regulation of membrane function by tyrosine phosphorylation. Genistein, in micromolar concentrations, alters the function of numerous ion channels and other membrane proteins, but only in few cases has it been demonstrated that the changes in membrane protein (ion channel) function are due to changes in a protein's phosphorylation status. The major common denominator characterizing proteins that are modulated by genistein seems to be that they are imbedded into, and span, the bilayer component of the plasma membrane. We therefore explored whether genistein could alter ion channel function by a bilayer-mediated mechanism and examined genistein's effect on gramicidin A (gA) channels in planar phospholipid bilayers. gA channels form by transmembrane dimerization of two nonconducting subunits, and genistein potentiates gA channel activity by increasing the appearance rate and prolonging the lifetime of bilayer-spanning gA dimers. That is, genistein shifts the equilibrium between nonconducting monomers and conducting dimers in favor of the bilayer-spanning dimers; the changes in channel activity therefore cannot be due to changes in bilayer fluidity. To obtain further insights into the mechanism underlying this modulation of gA channel function, we examined the effects of genistein on channels formed by gA analogues that differ in amino acid sequence. For a given channel length, the effects of genistein on gA dimerization do not depend on the specific sequence, or the chirality, of the channel-forming gA analogues. In contrast, when we change the channel length (by decreasing or increasing the number of amino acid residues in the sequence), or the bilayer thickness (by changing methylene groups in the acyl chains), the magnitude of genistein's effect increases with increasing hydrophobic mismatch between the channel length and the bilayer thickness. These results strongly suggest that genistein alters bilayer mechanical properties, which in turn modulates channel function. This bilayer-mediated mechanism is likely to apply to other pharmacological reagents and membrane proteins.

Amino Acid Sequence↗
PubMed2003

Structure of gramicidin a in a lipid bilayer environment determined using molecular dynamics simulations and solid-state NMR data.

Two different high-resolution structures recently have been proposed for the membrane-spanning gramicidin A channel: one based on solid-state NMR experiments in oriented phospholipid bilayers (Ketchem, R. R.; Roux, B.; Cross, T. A. Structure 1997, 5, 1655-1669; Protein Data Bank, PDB:1MAG); and one based on two-dimensional NMR in detergent micelles (Townsley, L. E.; Tucker, W. A.; Sham, S.; Hinton, J. F. Biochemistry 2001, 40, 11676-11686; PDB:1JNO). Despite overall agreement, the two structures differ in peptide backbone pitch and the orientation of several side chains; in particular that of the Trp at position 9. Given the importance of the peptide backbone and Trp side chains for ion permeation, we undertook an investigation of the two structures using molecular dynamics simulation with an explicit lipid bilayer membrane, similar to the system used for the solid-state NMR experiments. Based on 0.1 micros of simulation, both backbone structures converge to a structure with 6.25 residues per turn, in agreement with X-ray scattering, and broad agreement with SS backbone NMR observables. The side chain of Trp 9 is mobile, more so than Trp 11, 13, and 15, and undergoes spontaneous transitions between the orientations in 1JNO and 1MAG. Based on empirical fitting to the NMR results, and umbrella sampling calculations, we conclude that Trp 9 spends 80% of the time in the 1JNO orientation and 20% in the 1MAG orientation. These results underscore the utility of molecular dynamics simulations in the analysis and interpretation of structural information from solid-state NMR.

Anti-Bacterial Agents↗
PubMed2003

Hydrophobic coupling of lipid bilayer energetics to channel function.

The hydrophobic coupling between membrane-spanning proteins and the lipid bilayer core causes the bilayer thickness to vary locally as proteins and other "defects" are embedded in the bilayer. These bilayer deformations incur an energetic cost that, in principle, could couple membrane proteins to each other, causing them to associate in the plane of the membrane and thereby coupling them functionally. We demonstrate the existence of such bilayer-mediated coupling at the single-molecule level using single-barreled as well as double-barreled gramicidin channels in which two gramicidin subunits are covalently linked by a water-soluble, flexible linker. When a covalently attached pair of gramicidin subunits associates with a second attached pair to form a double-barreled channel, the lifetime of both channels in the assembly increases from hundreds of milliseconds to a hundred seconds--and the conductance of each channel in the side-by-side pair is almost 10% higher than the conductance of the corresponding single-barreled channels. The double-barreled channels are stabilized some 100,000-fold relative to their single-barreled counterparts. This stabilization arises from: first, the local increase in monomer concentration around a single-barreled channel formed by two covalently linked gramicidins, which increases the rate of double-barreled channel formation; and second, from the increased lifetime of the double-barreled channels. The latter result suggests that the two barrels of the construct associate laterally. The underlying cause for this lateral association most likely is the bilayer deformation energy associated with channel formation. More generally, the results suggest that the mechanical properties of the host bilayer may cause the kinetics of membrane protein conformational transitions to depend on the conformational states of the neighboring proteins.

Circular Dichroism↗
PubMed2002

Conformational control through translocational regulation: a new view of secretory and membrane protein folding.

We suggest a new view of secretory and membrane protein folding that emphasizes the role of pathways of biogenesis in generating functional and conformational heterogeneity. In this view, heterogeneity results from action of accessory factors either directly binding specific sequences of the nascent chain, or indirectly, changing the environment in which a particular domain is synthesized. Entrained by signaling pathways, these variables create a combinatorial set of necessary-but-not-sufficient conditions that enhance synthesis and folding of particular alternate, functional, conformational forms. We therefore propose that protein conformation is productively regulated by the cell during translocation across the endoplasmic reticulum (ER), a concept that may account for currently poorly understood aspects of physiological function, natural selection, and disease pathogenesis.

Animals↗