Jonas S. Johansson

Binding of the Volatile Anesthetic Chloroform to Albumin Demonstrated Using Tryptophan Fluorescence Quenching

The site(s) of action of the volatile general anesthetics remain(s) controversial, but evidence in favor of specific protein targets is accumulating. The techniques to measure directly volatile anesthetic binding to proteins are still under development. Further experience with the intrinsic protein fluorescence quenching approach to monitor anesthetic-protein complexation is reported using chloroform. Chloroform quenches the steady-state tryptophan fluorescence of bovine serum albumin (BSA) in a concentration-dependent, saturable manner with aK d = 2.7 ± 0.2 mm. Tryptophan fluorescence lifetime analysis reveals that the majority of the quenching is due to a static mechanism, indicative of anesthetic binding. The ability of chloroform to quench BSA tryptophan fluorescence was decreased markedly in the presence of 50% 2,2,2-trifluoroethanol, which causes loss of tertiary structural contacts in BSA, indicating that protein conformation is crucial for anesthetic binding. Circular dichroism spectroscopy revealed no measurable effect of chloroform on the secondary structure of BSA. The results suggest that chloroform binds to subdomains IB and IIA in BSA, each of which contains a single tryptophan. Earlier work has shown that these sites are also occupied by halothane. The present study therefore provides experimental support for the theory that structurally distinct general anesthetics may occupy the same domains on protein targets.

Binding of Halothane to Serum Albumin Demonstrated Using Tryptophan Fluorescence

Background : The site of action of general anesthesia remains controversial, but evidence in favor of specific protein target(s) is accumulating. Saturable binding of halothane to bovine serum albumin (BSA) has recently been reported using photoaffinity labeling and fluorine 19 nuclear magnetic resonance spectroscopy. We report a new approach to study anesthetic binding to soluble proteins, based on native tryptophan fluorescence. Methods : Thymol-free halothane and fatty acid-free BSA were equilibrated in gas-tight Hamilton syringes and dispensed into stoppered quartz cuvettes at predetermined dilutions. Steady-state fluorescence spectroscopy was used to study their interaction. Results : Halothane quenched the tryptophan fluorescence of BSA in a concentration-dependent, saturable manner with a dissociation constant = 1.8 ± 0.2 mM and a Hill number = 1.0 ± 0.1. The two optical isomers of halothane bound to BSA with equal affinity. The ability of halothane to quench BSA tryptophan fluorescence was markedly decreased at pH 3.0 (which causes full uncoiling of BSA), with loss of saturable binding. Diethyl ether displaced a portion of halothane from its binding sites. Circular dichroism spectroscopy revealed no significant effect of halothane or diethyl ether on the secondary structure of BSA. Conclusions : The results suggest that halothane binds in hydrophobic domains containing tryptophan in BSA. This approach may prove useful for studying the interaction of volatile anesthetics and proteins and has the advantage that the location of halothane in the protein is identified.

On the Relevance of “clinically Relevant Concentrations” of Inhaled Anesthetics in In Vitro Experiments

THE use of the phrase, “clinically relevant anesthetic concentrations” has become so enshrined in in vitro research on anesthetic action that its importance has assumed a prominent and rarely questioned status. To review, the yardstick by which most studies of anesthetic action are measured is sensitivity, or whether significant effects on in vitro systems can be measured at concentrations of general anesthetics used in people. Our contemporary inhalational anesthetics, halothane, isoflurane, and sevoflurane, produce aqueous concentrations of approximately 0.3 mM at 1 minimum alveolar concentration (MAC) and at 37°C. Thus, the logic goes, for an in vitro preparation or some other model system, to be in any way clinically relevant, it too must be affected, preferably by 50% of some maximal response at the same concentration. There are several implicit, but rarely discussed, assumptions underlying this logic, which we will consider in the following paragraphs. Most in vitro responses to a drug or ligand are nonlinear and, when plotted against the log of concentration, fit a sigmoid relation (Hill or logistic equation) reasonably well. Such relations are characterized as being “saturable,” meaning that progressively smaller increases in the target response occur with increases in ligand concentration beyond a specific point. If this in vitro response directly controls some behavior of an organism, one might also expect that behavior to reach a maximum, or “ceiling,” as the administered dose of the drug is increased. But is anesthesia saturable? It depends on how we define a state that we know very little about. The popular operational definition is the absence of a motor response to a noxious stimulus in an unparalyzed animal. This is known also as the MAC response (minimum alveolar concentration of inhaled anesthetic to prevent this response in 50% of a population). This is a categoric (binomial in this case) definition, and any such definition of necessity results in a saturable response. However, does an arbitrary behavioral definition require that the underlying central nervous system (CNS) events be saturable? Although the MAC response is a relatively clear behavioral endpoint, in terms of CNS function it is considerably less clear. We know that all noxious stimuli are not equal and that the dose–response curve for a simple skin incision, for example, is different from that describing intubation of the trachea. That different CNS functions have different sensitivities is also shown by the existence of the MAC-awake, and MAC-bar values. Further, no discrete electroencephalographic changes have been identified as indicating the achievement of MAC concentrations. Brain electrical activity becomes progressively depressed until approximately 3 MAC (approaching 1 mM aqueous concentration) for many of our agents until cardiovascular depression becomes critical. If cardiovascular and respiratory components are removed, as in, for example, cell culture, reversible influences of these compounds can be measured well above 3 MAC. Such progressive actions of anesthetics speak against finding a saturable CNS action that underlies binomial responses exemplified by MAC, and are more likely explained by progressive, simultaneous actions at many targets of comparable sensitivity. Alternatively, progressive CNS dysfunction could result from a more layered * Austin Lamont Associate Professor of Anesthesia, Departments of Anesthesia and Physiology.

A Designed Four-α-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.

First and Second Generation γ-Secretase Modulators (GSMs) Modulate Amyloid-β (Aβ) Peptide Production through Different Mechanisms

Background: γ-Secretase modulators (GSMs) hold potential as disease modifiers in Alzheimer disease; however, their mechanism of action is not completely understood. Results: Second generation in vivo active GSMs were described and shown to modulate Aβ production via a non-APP targeting mechanism, different from the NSAIDs class of GSMs. Conclusion: A growing class of second generation GSMs appears to target γ-secretase and displays a different mechanism of action compared with first generation GSMs. Significance: The identification of in vivo active non-APP targeting second generation GSMs may facilitate the development of novel therapeutics against AD. γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-β (Aβ) peptides. The Aβ42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs), such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide, have been suggested to modulate the Alzheimer-related Aβ production by targeting the APP. Here, we describe novel GSMs that are selective for Aβ modulation and do not impair processing of Notch, EphB2, or EphA4. The GSMs modulate Aβ both in cell and cell-free systems as well as lower amyloidogenic Aβ42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM, E2012, but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a 3H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined, these data provide compelling evidence of a growing class of in vivo active GSMs, which are selective for Aβ modulation and have a different mechanism of action compared with the original class of GSMs described.

Bound volatile general anesthetics alter both local protein dynamics and global protein stability.

BACKGROUND Recent studies have demonstrated that volatile general anesthetic agents such as halothane and isoflurane may bind to discrete sites on protein targets. In the case of bovine serum albumin, the sites of halothane and chloroform binding have been identified as being located in the IB and IIA subdomains. This structural information provides a foundation for more detailed studies into the potential mechanisms of anesthetic action. METHODS The effect of halothane and isoflurane and the nonimmobilizer 1,2-dichlorohexafluorocyclobutane on the mobility of the indole ring in the tryptophan residues of albumin was investigated using measurements of fluorescence anisotropy. Myoglobin served as a negative control. In addition, the effect of bound anesthetic agents on global protein stability was determined by thermal denaturation experiments using near-ultraviolet circular dichroism spectroscopy. RESULTS The fluorescence anisotropy measurements showed that halothane and isoflurane decreased the mobility of the indole rings in a concentration-dependent manner. The calculated dissociation constants were 1.6+/-0.4 and 1.3+/-0.3 mM for isoflurane and halothane, respectively. In contrast, both agents failed to increase the fluorescence anisotropy of the tryptophan residues in myoglobin, compatible with lack of binding. The nonimmobilizer 1,2-dichlorohexafluorocyclobutane caused no change in the fluorescence anisotropy of albumin. Binding of the anesthetic agents stabilized the native folded form of albumin to thermal denaturation. Analysis of the thermal denaturation data yielded dissociation constant values of 0.98+/-0.10 mM for isoflurane and 1.0+/-0.1 mM for halothane. CONCLUSIONS Attenuation of local side-chain dynamics and stabilization of folded protein conformations may represent fundamental modes of action of volatile general anesthetic agents. Because protein activity is crucially dependent on inherent flexibility, anesthetic-induced stabilization of certain protein conformations may explain how these important clinical agents change protein function.

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-alpha-helix bundle, (Aalpha(2)-L1M/L38M)(2), was designed to contain a long hydrophobic core, enclosed by four amphipathic alpha-helices, for specific anesthetic binding. The structural and dynamical analyses of (Aalpha(2)-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 (Aalpha(2)-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 A (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.

An Isothermal Titration Calorimetry Study on the Binding of Four Volatile General Anesthetics to the Hydrophobic Core of a Four-α-Helix Bundle Protein

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic-protein complexes. Previous work has demonstrated that the halogenated alkane volatile anesthetics halothane and chloroform bind to the hydrophobic core of the four-alpha-helix bundle (Aalpha(2)-L38M)(2) (Johansson et al., 2000, 2003). This study shows that the halogenated ether anesthetics isoflurane, sevoflurane, and enflurane are also bound to the hydrophobic core of the four-alpha-helix bundle, using isothermal titration calorimetry. Isoflurane and sevoflurane both bound to the four-alpha-helix bundle with K(d) values of 140 +/- 10 micro M, whereas enflurane bound with a K(d) value of 240 +/- 10 micro M. The DeltaH degrees values associated with isoflurane, sevoflurane, and enflurane binding were -7.7 +/- 0.1 kcal/mol, -8.2 +/- 0.2 kcal/mol, and -7.2 +/- 0.1 kcal/mol, respectively. The DeltaS degrees values accompanying isoflurane, sevoflurane, and enflurane binding were -8.5 cal/mol K, -10.4 cal/mol K, and -8.0 cal/mol K, respectively. The results indicate that the hydrophobic core of (Aalpha(2)-L38M)(2) is able to accommodate three modern ether anesthetics with K(d) values that approximate their clinical EC(50) values. The DeltaH degrees values point to the importance of polar interactions for volatile general anesthetic binding, and suggest that hydrogen bonding to the ether oxygens may be operative.

Binding of the volatile general anesthetics halothane and isoflurane to a mammalian β‐barrel protein

A molecular understanding of volatile anesthetic mechanisms of action will require structural descriptions of anesthetic–protein complexes. Porcine odorant binding protein is a 157 residue member of the lipocalin family that features a large β‐barrel internal cavity (515 ± 30 Å3) lined predominantly by aromatic and aliphatic residues. Halothane binding to the β‐barrel cavity was determined using fluorescence quenching of Trp16, and a competitive binding assay with 1‐aminoanthracene. In addition, the binding of halothane and isoflurane were characterized thermodynamically using isothermal titration calorimetry. Hydrogen exchange was used to evaluate the effects of bound halothane and isoflurane on global protein dynamics. Halothane bound to the cavity in the β‐barrel of porcine odorant binding protein with dissociation constants of 0.46 ± 0.10 mm and 0.43 ± 0.12 mm determined using fluorescence quenching and competitive binding with 1‐aminoanthracene, respectively. Isothermal titration calorimetry revealed that halothane and isoflurane bound with Kd values of 80 ± 10 µm and 100 ± 10 µm, respectively. Halothane and isoflurane binding resulted in an overall stabilization of the folded conformation of the protein by −0.9 ± 0.1 kcal·mol−1. In addition to indicating specific binding to the native protein conformation, such stabilization may represent a fundamental mechanism whereby anesthetics reversibly alter protein function. Because porcine odorant binding protein has been successfully analyzed by X‐ray diffraction to 2.25 Å resolution [ 1 ], this represents an attractive system for atomic‐level structural studies in the presence of bound anesthetic. Such studies will provide much needed insight into how volatile anesthetics interact with biological macromolecules.

Minimum structural requirement for an inhalational anesthetic binding site on a protein target

The present study makes use of direct photoaffinity labeling and fluorescence and circular dichroism spectroscopy to examine the interaction of the inhalational anesthetic halothane with the uncharged alpha-helical form of poly(L-lysine) over a range of chain lengths. Halothane bound specifically to long chain homopolymers (190 to 1060 residues), reaching a stable stoichiometry of 1 halothane to 160 lysine residues in polymers longer than 300 residues. Halothane bound only non-specifically to an alpha-helical 30 residue polymer and to all of the polymers in their charged, random coil form. The data suggest that halothane binding is a function of supersecondary structure whereby intramolecular helix-helix clusters form in the longer polymers, resulting in the creation of confined hydrophobic domains. Circular dichroism spectroscopy cannot demonstrate changes in poly(L-lysine) secondary structure at any chain length with up to 12 mM halothane, suggesting that extensive hydrogen bond disruption by the anesthetic does not occur.