Graphic representation of the results of kinetic analyses.
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Algorithms↗
Biomedical subjects
O S Andersen
Publications and source records attributed to O S Andersen.
Gramicidin channel kinetics under tension.
We have measured the effect of tension on dimerization kinetics of the channel-forming peptide gramicidin A. By aspirating large unilamellar vesicles into a micropipette electrode, we are able to simultaneously monitor membrane tension and electrical activity. We find that the dimer formation rate increases by a factor of 5 as tension ranges from 0 to 4 dyn/cm. The dimer lifetime also increases with tension. This behavior is well described by a phenomenological model of membrane elasticity in which tension modulates the mismatch in thickness between the gramicidin dimer and membrane.
Dimerization↗
Energetics of inclusion-induced bilayer deformations.
The material properties of lipid bilayers can affect membrane protein function whenever conformational changes in the membrane-spanning proteins perturb the structure of the surrounding bilayer. This coupling between the protein and the bilayer arises from hydrophobic interactions between the protein and the bilayer. We analyze the free energy cost associated with a hydrophobic mismatch, i.e., a difference between the length of the protein's hydrophobic exterior surface and the average thickness of the bilayer's hydrophobic core, using a (liquid-crystal) elastic model of bilayer deformations. The free energy of the deformation is described as the sum of three contributions: compression-expansion, splay-distortion, and surface tension. When evaluating the interdependence among the energy components, one modulus renormalizes the other: e.g., a change in the compression-expansion modulus affects not only the compression-expansion energy but also the splay-distortion energy. The surface tension contribution always is negligible in thin solvent-free bilayers. When evaluating the energy per unit distance (away from the inclusion), the splay-distortion component dominates close to the bilayer/inclusion boundary, whereas the compression-expansion component is more prominent further away from the boundary. Despite this complexity, the bilayer deformation energy in many cases can be described by a linear spring formalism. The results show that, for a protein embedded in a membrane with an initial hydrophobic mismatch of only 1 A, an increase in hydrophobic mismatch to 1.3 A can increase the Boltzmann factor (the equilibrium distribution for protein conformation) 10-fold due to the elastic properties of the bilayer.
Biophysical Phenomena↗
The conformational preference of gramicidin channels is a function of lipid bilayer thickness.
In order to understand how the material properties of lipid bilayers could affect integral membrane protein function, we examined the effect of a hydrophobic mismatch on the structure and function of membrane-spanning gramicidin channels. Changes in lipid bilayer thickness affect the conformational preference of membrane-spanning gramicidin A (gA) channels (single-stranded [SS] dimers <--> double-stranded [DS] dimers) and induces an additional conductance state in the standard (SS) beta6.3-helical channel. These results provide experimental evidence for the importance of energetic coupling between the bilayer and imbedded inclusions.
Amino Acid Sequence↗
Lipid bilayer electrostatic energy, curvature stress, and assembly of gramicidin channels.
Hydrophobic interactions between lipid bilayers and imbedded membrane proteins couple protein conformation to the mechanical properties of the bilayer. This coupling is widely assumed to account for the regulation of membrane protein function by the membrane lipids' propensity to form nonbilayer phases, which will produce a curvature stress in the bilayer. Nevertheless, there is only limited experimental evidence for an effect of bilayer curvature stress on membrane protein structure. We show that alterations in curvature stress, due to alterations in the electrostatic energy of dioleoylphosphatidylserine bilayers, modulate the structurally well-defined gramicidin A monomer <--> dimer reaction. Maneuvers that decrease the electrostatic energy of the unperturbed bilayer promote channel dissociation; we measure the change in interaction energy. The bilayer electrostatic energy thus can affect membrane protein structure by a mechanism that does not involve the electrostatic field across the bilayer, but rather electrostatic interactions among the phospholipid head groups in each monolayer which affect the bilayer curvature stress. These results provide further evidence for the importance of mechanical interactions between a bilayer and its imbedded proteins for protein structure and function.
Gramicidin↗
Gramicidin channels in phospholipid bilayers with unsaturated acyl chains.
In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.
Alkanes↗
The Journal of General Physiology.
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Molecular Biology↗
The 50th Annual Meeting and Symposium of the Society of General Physiologists. Cytoskeletal regulation of membrane function.
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Animals↗
Membrane stiffness and channel function.
Alterations in the stiffness of lipid bilayers are likely to constitute a general mechanism for modulation of membrane protein function. Gramicidin channels can be used as molecular force transducers to measure such changes in bilayer stiffness. As an application, we show that N-type calcium channel inactivation is shifted reversibly toward negative potentials by synthetic detergents that decrease bilayer stiffness. Cholesterol, which increases bilayer stiffness, shifts channel inactivation toward positive potentials. The voltage activation of the calcium channels is unaffected by the changes in stiffness. Changes in bilayer stiffness can be predicted from the molecular shapes of membrane-active compounds, which suggests a basis for the pharmacological effects of such compounds.
Calcium Channels↗
Gramicidin channels--a solvable membrane "protein" folding problem.
The linear gramicidins are peptide antibiotics that form cation-selective channels in lipid bilayers. Gramicidin channels have very well-defined functional characteristics, and the structure of membrane-spanning gramicidin A channels is known at atomic resolution. These features make the gramicidins well suited to study how the amino acid sequence encodes the structure and function of a membrane-spanning channel. We show how one can use electrophysiological measurements to obtain structural information about conducting channels and to quantify the conformational preferences of sequence-substituted gramicidin mutants.
Amino Acid Sequence↗
Gramicidin channel function does not depend on phospholipid chirality.
Chiral interactions are often important determinants for molecular recognition in chemistry and biochemistry. In order to determine whether the phospholipid backbone could be important for the conformational preference of membrane-spanning channels, we made use of the linear pentadecapeptide antibiotic gramicidin A (gA+) and a Trp-->Phe-substituted gA+ analogue, gramicidin M+ (gM+), as well as their enantiomers [gramicidin A- (gA-) and gramicidin M- (gM-), respectively]. All four analogues form conducting channels in planar bilayers formed from the dialkylphospholipids (R)- or (S)- dioleylphosphatidylcholine or from the diacylphospholipid (R)-dioleoylphosphatidylcholine. The characteristics of channels formed by the two gramicidin A enantiomers, or the two gramicidin M enantiomers, in membranes formed by either of the dioleylphosphatidylcholine enantiomers are indistinguishable. Similarly, channels formed by either pair of gramicidin enantiomers in dioleoylphosphatidylcholine bilayers are indistinguishable. We conclude that chiral interactions between gramicidin channels and the lipids in the host bilayer cannot be important determinants of gramicidin channel structure or function. The membrane/solution interface, therefore, seems to organize the channel structure because of the general characteristics of the nonpolar/polar transition at the interface rather than because of specific chemical interactions.
Amino Acid Sequence↗
Stabilizing effect of D-alanine2 in gramicidin channels.
We have investigated the effects of replacing Gly2 by D-Ala2 in gramicidin A (gA) analogues that have either L-Val, L-Ala, or Gly as the formyl-N-terminal residue. Circular dichroism, two-dimensional nuclear magnetic resonance, and hybrid channel experiments all show that [Ala1,D-Ala2]gA channels are structurally equivalent to the native [Val1,Gly2]gA channels, being formyl-NH-to-formyl-NH dimers of single-stranded, right-handed beta 6.3 helices. Replacing the Val1 of gA by Ala or Gly decreases the average channel duration. Replacing Gly2 by D-Ala in [Val1,Gly2]gA increases the average channel duration 4-fold and the single-channel conductance by approximately 15%; replacing Gly2 with D-Ala in [Ala1,Gly2]gA or [Gly1,Gly2]-gA leads in each case to a 10-fold increase in the average channel duration with only modest changes in the single-channel conductance, which depends on the identity of the position-one residue and the permeant ion. These results illustrate the importance of neighboring-residue side chain and backbone interactions for the modulation of channel properties.
Alanine↗
Voltage-dependent gating of an asymmetric gramicidin channel.
In an effort to understand the molecular mechanisms of voltage activation of ion channels, we have chosen a system of known structure and examined the properties of heterodimeric channels formed between [Val1]gramicidin A ([Val1]gA) and [F6Val1]gramicidin A ([F6Val1]gA). Gramicidin channels are usually not voltage-dependent; but the introduction of a single symmetry-breaking dipolar F6Val1 residue into a ([Val1]gA)2 dimer to form the [F6Val1]gA/[Val1]gA heterodimer induces voltage-dependent transitions between two conducting states: a high-conductance state and a zero conductance (closed) state. The distribution between these states varies as a function of the applied potential but is not dependent on the nature of the permeant ion (H+ or Cs+). The permeating ions do not seem to contribute to the apparent gating charge.
Calcium Channels↗
Helix sense of gramicidin channels as a "nonlocal" function of the primary sequence.
Gramicidin A (gA) channels are dimers formed by right-handed beta 6.3-helical monomers. The stereochemical basis for the preference of a right-handed conformation remains obscure, but it has earlier been demonstrated that the handedness can be shifted by changing the chirality of each residue in the LD-sequence and therefore is determined by the peptide itself and not by channel-membrane interactions. We now examine the contributions of Trp15, the central Val residues 6-8, and residues 1-5. None of these alone are sufficient to specify the helix sense. To examine the D-Val6-L-Val7-D-Val8 sequence, the register of the 3 valines was shifted by one to L-Val5-D-Val6-L-Val7. The resulting analogue, [Val5,D-Ala8]gA, forms channels with a conductance and duration that are both somewhat less than those of gA channels. The reduced channel duration can be attributed to a steric conflict between the side chains of Val1 in one monomer and Val5 in the other monomer. The helix handedness is not altered by this modification, as shown by circular dichroism and two-dimensional nuclear magnetic resonance spectroscopy and by hybrid channel experiments. [Val5,D-Ala8]gA forms hybrid channels with gA (which forms right-handed channels), but not with des-Val1-gA- (which forms left-handed channels). Similar hybrid channel analysis shows that des-Trp15-gA and [L-Ala1,D-Ala2,L-Ala3,D-Ala4]gA also form right-handed channels. We conclude that the helix handedness most probably is a complex function of the arrangement of both the D-Val-L-Val-D-Val and the L-Trp-(D-Leu-L-Trp)3 segments.
Amino Acid Sequence↗
Closed state of gramicidin channel detected by X-ray in-plane scattering.
An analogue of gramicidin A (gA) was synthesized with the formyl group replaced by a BOC group. The analogue (BOC-gA) exhibited single channel conduction, but the channel is 5-order-of-magnitude destabilized relative to the gA channel. Hydrated mixtures of gramicidin and dilauroyl phosphatidylcholine in the molar ratio of 1:10 were prepared into uniformly aligned multiple bilayers, and X-ray scattering with the momentum transfer in the plane of the membrane was measured. Analysis with the help of computer simulations showed that 70% of BOC-gA are monomers. Thus for the first time it was shown that gramicidin monomers are stable inside the monolayers of a lipid membrane. Furthermore, the monomers have the same beta helical conformation as the dimeric channel. The result suggests the possibility that when a gramicidin channel is closed, it dissociates into two monomers floating in opposite monolayers.
Computer Simulation↗
Asymmetric gramicidin channels: heterodimeric channels with a single F6Val1 residue.
Substitution of Val1 by 4,4,4,4',4',4'-F6Val in [Val1]gramicidin A ([Val1]gA) produces channels in which the effects of amino acid replacements on dimer stability and ion permeation are nonadditive. If only one Val1 (in a symmetric [Val1]gA channel) is substituted by F6Val, the resulting heterodimeric channels are destabilized relative to both homodimeric parent channels and the single-channel conductance of the heterodimeric channels is reduced relative to the parent channels (Russell, E. W. B., L. B. Weiss, F. I. Navetta, R. E. Koeppe II, and O. S. Andersen. 1986. Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position #1 in gramicidin A. Biophys. J. 49:673; Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence. J. Mol. Biol. 211:221-234). To understand the basis for this destabilization, we have examined further the characteristics of [F6Val1]/[Xxx1]gA heterodimers, where Xxx = Gly, Val, and Ala. These heterodimeric channels show rapid current transitions between (at least) two current levels and display asymmetric i-V characteristics. The orientation of the heterodimers relative to the applied potential was determined by asymmetric addition of the gramicidin analogs, one to each side of a preformed bilayer. The current transitions are most clearly illustrated for [F6Val1]/[Gly1]gA heterodimers, which possess two finite and well defined current levels. Based on the existence of these two conductance states and the analysis of duration and interval distributions, we conclude that the transitions between the two current levels correspond to conformational transitions in "stable" heterodimers. In the case of [F6Val1]/[Val1]gA and [F6Val1]/[Ala1]gA heterodimers, the low-conductance state is indistinguishable from zero. The two (or more) conductance states presumably correspond to different orientations of the dipolar F6Val1 side chain. The distribution between the high- and the low-conductance states varies as a function of potential in [F6Val1]/[Gly1]gA channels. These characteristics cause the [F6Val1]/nonpolar (Val, Ala, Gly)gA hybrid channels to serve as a "simple" model for understanding gating transitions in membrane-spanning channels.
Amino Acid Sequence↗
Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers.
Lipid metabolites, free fatty acids and lysophospholipids, modify the function of membrane proteins including ion channels. Such alterations can occur through signal transduction pathways, but may also result from "direct" effects of the metabolite on the protein. To investigate possible mechanisms for such direct effects, we examined the alterations of gramicidin channel function by lysophospholipids (LPLs): lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylserine (LPS), and lysophosphatidylinositol (LPI). The experiments were done on planar bilayers formed by diphytanoylphosphatidylcholine in n-decane a system where receptor-mediated effects can be excluded. At aqueous concentrations below the critical micelle concentration (CMC), LPLs can increase the dimerization constant for membrane-bound gramicidin up to 500-fold (at 2 microM). The relative potency increases as a function of the size of the polar head group, but does not seem to vary as a function of head group charge. The increased dimerization constant results primarily from an increase in the rate constant for channel formation, which can increase more than 100-fold (in the presence of LPC and LPI), whereas the channel dissociation rate constant decreases only about fivefold. The LPL effect cannot be ascribed to an increased membrane fluidity, which would give rise to an increased channel dissociation rate constant. The ability of LPC to decrease the channel dissociation rate constant varies as a function of channel length (which is always less than the membrane's equilibrium thickness): as the channel length is decreased, the potency of LPC is increased. LPC has no effect on membrane thickness or the surface tension of monolayers at the air/electrolyte interface. The bilayer-forming glycerolmonooleate does not decrease the channel dissociation rate constant. These results show that LPLs alter gramicidin channel function by altering the membrane deformation energy, and that the changes in deformation energy can be related to the molecular "shape" of the membrane-modifying compounds. Similar alterations in the mechanical properties of biological membranes may form a general mechanism by which one can alter membrane protein function.
Amino Acid Sequence↗
Energetics of heterodimer formation among gramicidin analogues with an NH2-terminal addition or deletion. Consequences of missing a residue at the join in the channel.
We examined the properties of membrane-spanning channels formed by gramicidin analogues that differ from [Val1]gramicidin A by having a single residue deletion or insertion at the formyl-NH terminus and of hybrid channels formed between such 14-, 15-, and 16-residue analogues. The channels' backbone structure, and helix sense, are not affected by the sequence modifications, because hybrid channels were observed for all combinations tested, and there was no excess energetic cost associated with hybrid channel formation. When hybrid channels form between analogues of different length the hybrid channel stability depends on the nature of the sequence dissimilarity. If two analogues differ by one residue (delta n = 1), the hybrid channels are destabilized by approximately 10 kJ/mol, because there is a defect (a "gap" in the peptide backbone) at the join between the two beta 6.3-helical monomers such that the dimer is stabilized by only five intermolecular C = O ... H-N hydrogen bonds rather than the usual six. This defect also alters the hybrid channels' permeability characteristics: the single-channel conductances are decreased, as if there were an additional barrier to ion movement through the channel. If the formyl-NH-terminal residue is Gly (and delta n = 1), the hybrid channels show multi-state behavior with voltage-dependent transitions between two conductance levels. If two analogues differ by two residues (delta n = 2), the hybrid channels are stabilized by 3 kJ/mol, indicating that structural continuity at the join between the monomers has been restored, as have the hybrid channels' permeability characteristics. The increased hybrid channel stability (when delta n = 2) may arise from altered membrane-channel interactions.
Amino Acid Sequence↗