A designed four-alpha-helix bundle that binds the volatile general anesthetic halothane with high affinity

The structural features of volatile anesthetic binding sites on proteins are being examined with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work has suggested that introducing a cavity into the hydrophobic core improves anesthetic binding affinity. The more polarizable methionine side chain was substituted for a leucine, in an attempt to enhance the dispersion forces between the ligand and the protein. The resulting bundle variant has an improved affinity (K(d) = 0.20 +/- 0.01 mM) for halothane binding, compared with the leucine-containing bundle (K(d) = 0.69 +/- 0.06 mM). Photoaffinity labeling with (14)C-halothane reveals preferential labeling of the W15 residue in both peptides, supporting the view that fluorescence quenching by bound anesthetic reports both the binding energetics and the location of the ligand in the hydrophobic core. The rates of amide hydrogen exchange were similar for the two bundles, suggesting that differences in binding affinity were not due to changes in protein stability. Binding of halothane to both four-alpha-helix bundle proteins stabilized the native folded conformations. Molecular dynamics simulations of the bundles illustrate the existence of the hydrophobic core, containing both W15 residues. These results suggest that in addition to packing defects, enhanced dispersion forces may be important in providing higher affinity anesthetic binding sites. Alternatively, the effect of the methionine substitution on halothane binding energetics may reflect either improved access to the binding site or allosteric optimization of the dimensions of the binding pocket. Finally, preferential stabilization of folded protein conformations may represent a fundamental mechanism of inhaled anesthetic action.     

Molecular dynamics simulations of C2F6 effects on gramicidin A: implications of the mechanisms of general anesthesia

It was recently postulated that the effects of general anesthetics on protein global dynamics might underlie a unitary molecular mechanism of general anesthesia. To verify that the specific dynamics effects caused by general anesthetics were not shared by nonanesthetic molecules, two parallel 8-ns all-atom molecular dynamics simulations were performed on a gramicidin A (gA) channel in a fully hydrated dimyristoylphosphatidylcholine membrane in the presence and absence of hexafluoroethane (HFE), which structurally resembles the potent anesthetic molecule halothane but produces no anesthesia. Similar to halothane, HFE had no measurable effects on the gA channel structure. In contrast to halothane, HFE produced no significant changes in the gA channel dynamics. The difference between halothane and HFE on channel dynamics can be attributed to their distinctly different distributions within the lipid bilayer and consequently to the different interactions of the anesthetic and the nonanesthetic molecules with the channel-anchoring tryptophan residues. The study further supports the notion that anesthetic-induced changes in protein global dynamics may play an important role in mediating anesthetic actions on proteins.     

Expression and characterization of a four-alpha-helix bundle protein that binds the volatile general anesthetic halothane

The structural features of volatile anesthetic binding sites on proteins are being investigated with the use of a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. The current study describes the bacterial expression, purification, and initial characterization of the four-alpha-helix bundle (Aalpha(2)-L1M/L38M)(2). The alpha-helical content and stability of the expressed protein are comparable to that of the chemically synthesized four-alpha-helix bundle (Aalpha(2)-L38M)(2) reported earlier. The affinity for binding halothane is somewhat improved with a K(d) = 120 +/- 20 microM as determined by W15 fluorescence quenching, attributed to the L1M substitution. Near-UV circular dichroism spectroscopy demonstrated that halothane binding changes the orientation of the aromatic residues in the four-alpha-helix bundle. Nuclear magnetic resonance experiments reveal that halothane binding results in narrowing of the peaks in the amide region of the one-dimensional proton spectrum, indicating that bound anesthetic limits protein dynamics. This expressed protein should prove to be amenable to nuclear magnetic resonance structural studies on the anesthetic complexes, because of its relatively small size (124 residues) and the high affinities for binding volatile anesthetics. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules and will provide guidelines regarding the general architecture of binding sites on central nervous system proteins.     

G protein {beta}{gamma} gating confers volatile anesthetic inhibition to Kir3 channels

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the βγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gβγ available to the channels, we asked whether reducing available Gβγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gβγ using the C-terminal domain of β-adrenergic receptor kinase (cβARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gβγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gβγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gβγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.     

Influence of hydrophobic mismatch on structures and dynamics of gramicidin a and lipid bilayers

Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4–5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations—except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.     

The membrane interface dictates different anchor roles for "inner pair" and "outer pair" tryptophan indole rings in gramicidin A channels

We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded conformers. Within this context, the (2)H NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state (2)H NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe(13,15)]gA channels have Na(+) currents that are ~50% and lifetimes that are ~80% of those of native gA channels. The double-stranded conformer(s) of [Phe(9,11)]gA do not form detectable channels. The minor single-stranded population of [Phe(9,11)]gA forms channels with Na(+) currents that are ~25% and single-channel lifetimes that are ~300% of those of native gA channels. Our results suggest that Trp(9) and Trp(11), when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp(9) and Trp(11) are relatively more important for the single-channel conductance than are the dipoles of Trp(13) and Trp(15).     

The sensitivity of G protein-activated K+ channels toward halothane is essentially determined by the C terminus

G protein-activated K(+) channels (GIRKs or Kir3.x) are targets for the volatile anesthetic, halothane. When coexpressed with the m(2) acetylcholine (ACh) receptor in Xenopus oocytes, agonist-activated GIRK1(F137S)- and GIRK2-mediated currents are inhibited by halothane, whereas in the absence of ACh, high concentrations of halothane induce GIRK1(F137S)-mediated currents. To elucidate the molecular mechanism of halothane action on GIRK currents of different subunit compositions, we constructed deletion mutants of GIRK1(F137S) (GIRK1(Delta363*)) and GIRK2 (GIRK2(Delta356)) lacking the C-terminal ends, as well as chimeric GIRK channels. Mutated GIRK channels showed normal currents when activated by ACh but exhibited different pharmacological properties toward halothane. GIRK2(Delta356) showed no sensitivity against the inhibitory action of halothane but was activated by halothane in the absence of an agonist. GIRK1(Delta363*) was activated by halothane more efficiently. Currents mediated by chimeric channels were inhibited by anesthetic concentrations that were at least 30-fold lower than those necessary to decrease GIRK2 wild type currents. Glutathione S-transferase pulldown experiments did not show displacement of bound Gbetagamma by halothane, indicating that halothane does not interfere with Gbetagamma binding. Single channel experiments revealed an influence of halothane on the gating of the channels: The agonist-induced currents of GIRK1 and GIRK2, carried mainly by brief openings, were inhibited, whereas higher concentrations of the anesthetic promoted long openings of GIRK1 channels. Because the C terminus is crucial for these effects, an interaction of halothane with the channel seems to be involved in the mechanism of current modulation.     

Neighboring aliphatic/aromatic side chain interactions between residues 9 and 10 in gramicidin channels

The interactions between an aliphatic or phenyl side chain and an indole ring in a phospholipid environment were investigated by synthesizing and characterizing gramicidins in which Trp(9) was ring-labeled and D-Leu(10) was replaced by D-Val, D-Ala, or D-Phe. All three analogues form conducting channels, with conductances that are lower than that of gramicidin A (gA) channels. The channel lifetimes vary by less than 50% from that of gA channels. Circular dichroism spectra and size-exclusion chromatography show that the conformation of each analogue in dimyristoylphosphatidylcholine (DMPC) vesicles is similar to the right-handed beta(6.3)-helical conformation that is observed for gA. (2)H NMR spectra of oriented samples in DMPC show large changes for the Trp(9) ring when residue 10 is modified, suggesting a steric interaction between D-Leu(10) and Trp(9), in agreement with previous acylation studies (R. E. Koeppe II et al. (1995) Biochemistry 34, 9299-9307). The outer quadrupolar splitting for Trp(9) is unchanged with D-Phe(10), at approximately 153 kHz, but increases by approximately 25 kHz with D-Val(10) and decreases by approximately 10 kHz with D-Ala(10). With D-Ala(10) or D-Val(10), the outer resonance splits into two in a temperature-dependent manner. The NMR spectra indicate that the side chain torsion angles chi1 and chi2 for Trp(9) change when residue 10 is substituted. The changes in chi1 are small, in all cases less than 10 degrees, as is Deltachi2 when D-Ala(10) is introduced, but with D-Val(10) and D-Phe(10) Deltachi2 is at least 25 degrees. We conclude that D-Leu(10) helps to stabilize an optimal orientation of Trp(9) in gA channels in lipid bilayers and that changes in Trp orientation alter channel conductance and lifetime without affecting the basic channel fold.     

Molecular mapping of general anesthetic sites in a voltage-gated ion channel

Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces.     

Interaction of anesthetics with open and closed conformations of a potassium channel studied via molecular dynamics and normal mode analysis

A variety of experiments suggest that membrane proteins are important targets of anesthetic molecules, and that ion channels interact differently with anesthetics in their open and closed conformations. The availability of an open and a closed structural model for the KirBac1.1 potassium channel has made it possible to perform a comparative analysis of the interactions of anesthetics with the same channel in its open and closed states. To this end, all-atom molecular dynamics simulations supplemented by normal mode analysis have been employed to probe the interactions of the inhalational anesthetic halothane with both an open and closed conformer of KirBac1.1 embedded in a lipid bilayer. Normal mode analysis on the closed and open channel, in the presence and absence of halothane, reveals that the anesthetic modulates the global as well as the local dynamics of both conformations differently. In the case of the open channel, the observed reduction of flexibility of residues in the inner helices suggests a functional modification action of anesthetics on ion channels. In this context, preferential quenching of the aromatic residue motion and modulation of global dynamics by halothane may be seen as steps toward potentiating or favoring open state conformations. These molecular dynamics simulations provide the first insights into possible specific interactions between anesthetic molecules and ion channels in different conformations.     

Four-α-Helix Bundle with Designed Anesthetic Binding Pockets. Part II: Halothane Effects on Structure and Dynamics

As a model of the protein targets for volatile anesthetics, the dimeric four-α-helix bundle, (Aα₂-L1M/L38M)₂, was designed to contain a long hydrophobic core, enclosed by four amphipathic α-helices, for specific anesthetic binding. The structural and dynamical analyses of (Aα₂-L1M/L38M)₂ in the absence of anesthetics (another study) showed a highly dynamic antiparallel dimer with an asymmetric arrangement of the four helices and a lateral accessing pathway from the aqueous phase to the hydrophobic core. In this study, we determined the high-resolution NMR structure of (Aα₂-L1M/L38M)₂ in the presence of halothane, a clinically used volatile anesthetic. The high-solution NMR structure, with a backbone root mean-square deviation of 1.72 Å (2JST), and the NMR binding measurements revealed that the primary halothane binding site is located between two side-chains of W15 from each monomer, different from the initially designed anesthetic binding sites. Hydrophobic interactions with residues A44 and L18 also contribute to stabilizing the bound halothane. Whereas halothane produces minor changes in the monomer structure, the quaternary arrangement of the dimer is shifted by about half a helical turn and twists relative to each other, which leads to the closure of the lateral access pathway to the hydrophobic core. Quantitative dynamics analyses, including Modelfree analysis of the relaxation data and the Carr-Purcell-Meiboom-Gill transverse relaxation dispersion measurements, suggest that the most profound anesthetic effect is the suppression of the conformational exchange both near and remote from the binding site. Our results revealed a novel mechanism of an induced fit between anesthetic molecule and its protein target, with the direct consequence of protein dynamics changing on a global rather than a local scale. This mechanism may be universal to anesthetic action on neuronal proteins.     

Anesthetic Binding in a Pentameric Ligand-Gated Ion Channel: GLIC

Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. An additional binding site at the EC-TM interface was predicted by docking analysis and validated by quenching experiments on the N200W GLIC mutant. The binding affinities (K_D) of 2.3 ± 0.1 mM and 0.10 ± 0.01 mM were derived from the fluorescence quenching data of halothane and thiopental, respectively. Docking these anesthetics to the original GLIC crystal structure and the structures relaxed by molecular dynamics simulations revealed intrasubunit sites for most halothane binding and intersubunit sites for thiopental binding. Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.     

Molecular dynamics simulation of a synthetic four-alpha-helix bundle that binds the anesthetic halothane.

The structural features of binding sites for volatile anesthetics are examined by performing a molecular dynamics simulation study of the synthetic four-alpha-helix bundles (Aalpha2)2, which are formed by association of two 62-residue di-alpha-helical peptides. The peptide bundle (Aalpha2)2 was designed by Johansson et al. [Biochemistry 37 (1998) 1421-1429] and was shown experimentally to have a high affinity for the binding of the anesthetic halothane (CF3CBrCIH) in a hydrophobic cavity. Since (Aalpha2)2 can exhibit either the anti or syn topologies, the two distinct bundles are simulated both in the presence and in the absence of halothane. Nanosecond length molecular dynamics trajectories were generated for each system at room temperature (T = 298 K). The structural and dynamic effects of the inclusion of halothane are compared, illustrating that the structures are stable over the course of the simulation; that the (Aalpha2)2 bundles have suitable pockets that can accommodate halothane; that the halothane remains in the designed hydrophobic cavity in close proximity to the Trp residues with a preferred orientation; and that the dimensions of the peptide are perturbed by the inclusion of an anesthetic molecule.     

Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action

TASK-1 and TASK-3, members of the two-pore-domain channel family, are widely expressed leak potassium channels responsible for maintenance of cell membrane potential and input resistance. They are sites of action for a variety of modulatory agents, including volatile anesthetics and neurotransmitters/hormones, the latter acting via mechanisms that have remained elusive. To clarify these mechanisms, we generated mutant channels and found that alterations disrupting anesthetic (halothane) activation of these channels also disrupted transmitter (thyrotropin-releasing hormone, TRH) inhibition and did so to a similar degree. For both TASK-1 and TASK-3, mutations (substitutions with corresponding residues from TREK-1) in a six-residue sequence at the beginning of the cytoplasmic C terminus virtually abolished both anesthetic activation and transmitter inhibition. The only sequence motif identified with a classical signaling mechanism in this region is a potential phosphorylation site; however, mutation of this site failed to disrupt modulation. TASK-1 and TASK-3 differed insofar as a large portion of the C terminus was necessary for the full effects of halothane and TRH on TASK-3 but not on TASK-1. Finally, tandem-linked TASK-1/TASK-3 heterodimeric channels were fully modulated by anesthetic and transmitter, and introduction of the identified mutations either into the TASK-1 or the TASK-3 portion of the channel was sufficient to disrupt both effects. Thus, both anesthetic activation and transmitter inhibition of these channels require a region at the interface between the final transmembrane domain and the cytoplasmic C terminus that has not been associated previously with receptor signal transduction. Our results also indicate a close molecular relationship between these two forms of modulation, one endogenous and the other clinically applied.     

Low-affinity analytical chromatography for measuring inhaled anesthetic binding to isolated proteins

The direct measure of volatile anesthetic binding to protein is complicated by weak affinity and therefore rapid kinetics. Consequently, several puted targets for these clinically important drugs have only functional data to support a direct mode of action. While several methods for measuring some aspects of binding are available, all have significant limitations. We introduce the use of analytical chromatography for the purpose of directly measuring volatile anesthetic binding to protein, and show that it can provide estimates of both affinity and stoichiometry for proteins that can be obtained in fairly high purity and mass. Using this approach we characterize halothane binding to serum albumin as low affinity and multisite, and to myoglobin or cytochrome C as strictly nonspecific. This approach will be useful in directly characterizing equilibrium, solution binding to isolated proteins in preparation for more time-consuming methods with structural resolution.     

Anesthetic and ethanol effects on spontaneously opening glycine receptor channels

Strychnine-sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand-gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor alpha1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1-trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand-gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on alpha1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding.     

Role of aromatic side chains in the binding of volatile general anesthetics to a four-alpha-helix bundle

Currently, the mechanism by which anesthesia occurs is thought to involve the direct binding of inhaled anesthetics to ligand-gated ion channels. This hypothesis is being studied using four-alpha-helix bundles as model systems for the transmembrane domains of the natural "receptor" proteins. This study concerns the role in anesthetic binding played by aromatic side chains in the binding cavity of a four-alpha-helix bundle designed to assume a Rop-like fold. Specifically, the effect of the substitution W15Y on bundle structure, stability, and anesthetic binding energetics was investigated. No appreciable effect of substituting W for Y on the secondary structure or the thermodynamic stability of the four-alpha-helix bundle was identified. However, the substitution W15Y resulted in about 6- and 3-fold decreases in halothane and chloroform binding affinities, respectively. This effect may reflect weaker dipole-aromatic quadrupole interactions between the aromatic side chain and the anesthetic in the tyrosine-containing species, which possesses the smaller aromatic ring system. For these anesthetic binding proteins, this class of interaction occurs when the permanent nonspherical distribution of electrons in the aromatic ring systems interact with the weakly acidic CH group of the anesthetics.     

Interaction of anesthetics with the Rho GTPase regulator Rho GDP dissociation inhibitor

The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.     

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, III: Molecular Dynamics Simulation Incorporating a Cyanophenylalanine Spectroscopic Probe

A nitrile-derived amino acid, Phe_CN, has been used as an internal spectroscopic probe to study the binding of an inhalational anesthetic to a model membrane protein. The infrared spectra from experiment showed a blue-shift of the nitrile vibrational frequency in the presence of the anesthetic halothane. To interpret the infrared results and explore the nature of the interaction between halothane and the model protein, all-atom molecular dynamics (MD) simulations have been used to probe the structural and dynamic properties of the protein in the presence and absence of one halothane molecule. The frequency shift analyzed from MD simulations agrees well with the experimental infrared results. Decomposition of the forces acting on the nitrile probes demonstrates an indirect impact on the probes from halothane, namely a change of the protein's electrostatic local environment around the probes induced by halothane. Although the halothane remains localized within the designed hydrophobic binding cavity, it undergoes a significant amount of translational and rotational motion, modulated by the interaction of the trifluorine end of halothane with backbone hydrogens of the residues forming the cavity. This dominant interaction between halothane and backbone hydrogens outweighs the direct interaction between halothane and the nitrile groups, making it a good “spectator” probe of the halothane-protein interaction. These MD simulations provide insight into action of anesthetic molecules on the model membrane protein, and also support the further development of nitrile-labeled amino acids as spectroscopic probes within the designed binding cavity.