A calorimetric study on the binding of six general anesthetics to the hydrophobic core of a model protein

Zhang, Tao; Johansson, Jonas

doi:10.1016/j.bpc.2004.08.009

Cited by 8 publications

(5 citation statements)

References 25 publications

(45 reference statements)

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“…1,5 Isothermal titration calorimetry revealed that the halogenated ether anesthetics isoflurane, enflurane, sevoflurane, and desflurane also bound to the hydrophobic core of the four-R-helix bundle (AR 2 -L38M) 2 with dissociation constants that mirror their clinical EC 50 values. 6,7 These studies indicate that the four-R-helix bundle (AR 2 -L38M) 2 represents a reasonable model for the in vivo central nervous system sites of anesthetic action.…”

Section: Introductionmentioning

confidence: 93%

“…Previous work using fluorescence quenching has demonstrated that the halogenated alkane volatile anesthetics halothane and chloroform bind to the designed packing defect in the hydrophobic core of the chemically synthesized four-α-helix bundle (Aα 2 -L38M) 2 with dissociation constants that are comparable to their clinical EC 50 values. , Isothermal titration calorimetry revealed that the halogenated ether anesthetics isoflurane, enflurane, sevoflurane, and desflurane also bound to the hydrophobic core of the four-α-helix bundle (Aα 2 -L38M) 2 with dissociation constants that mirror their clinical EC 50 values. , These studies indicate that the four-α-helix bundle (Aα 2 -L38M) 2 represents a reasonable model for the in vivo central nervous system sites of anesthetic action.…”

Section: Introductionmentioning

confidence: 99%

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Expression and Characterization of a Four-α-Helix Bundle Protein That Binds the Volatile General Anesthetic Halothane

et al. 2005

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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.

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Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Expression and Characterization of a Four-α-Helix Bundle Protein That Binds the Volatile General Anesthetic Halothane

et al. 2005

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“…To overcome these obstacles, small, water-soluble four-α-helix bundle proteins have been designed, produced, and used as well-defined model systems to explore how inhaled anesthetics bind to proteins ( − ). Each four-α-helix bundle protein is a dimer of two 27-residue α-helices joined by an 8-residue glycine linker (Figure ).…”

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confidence: 99%

Kinetics of Anesthetic-Induced Conformational Transitions in a Four-α-Helix Bundle Protein

2006

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Inhaled anesthetics are thought to alter the conformational states of Cys-loop ligand-gated ion channels (LGICs) by binding within discrete cavities that are lined by portions of four α-helical transmembrane domains. Because Cys-loop LGICs are complex molecules that are notoriously difficult to express and purify, scaled-down models have been used to better understand the basic molecular mechanisms of anesthetic action. In this study, stopped-flow fluorescence spectroscopy was used to define the kinetics with which inhaled anesthetics interact with (Aα 2 -L1M/L38M) 2 , a four-α-helix bundle protein that was designed to model anesthetic binding sites on Cys-loop LGICs. Stopped-flow fluorescence traces obtained upon mixing (Aα 2 -L1M/L38M) 2 with halothane revealed immediate, fast, and slow components of quenching. The immediate component, which occurred within the mixing time of the spectrofluorimeter, was attributed to direct quenching of tryptophan fluorescence upon halothane binding to (Aα 2 -L1M/L38M) 2 . This was followed by a bi-exponential fluorescence decay containing fast and slow components, reflecting anesthetic-induced conformational transitions. Fluorescence traces obtained in studies using sevoflurane, isoflurane, and desflurane, which poorly quench tryptophan fluorescence, did not contain the immediate component. However, these anesthetics did produce the fast and slow components, indicating that they also alter the conformation of (Aα 2 -L1M/L38M) 2 . Cyclopropane, an anesthetic which acts with unusually low potency on Cys-loop LGICs, acted with low apparent potency on (Aα 2 -L1M/L38M) 2 . These results suggest that four-α-helix bundle proteins may be useful models of in-vivo sites of action that allow use of a wide range of techniques to better understand how anesthetic binding leads to changes in protein structure and function. KeywordsAnesthesia; Cyclopropane; Desflurane; Fluorescence; Halothane; Isoflurane; Kinetics; Sevoflurane; Spectroscopy; Stopped-flow Inhaled general anesthetics alter the function of numerous proteins (1-5). Although the receptor site(s) responsible for producing anesthesia are not known with certainty, members of an anesthetic-sensitive superfamily of homologous Cys-loop ligand-gated ion channels (LGICs) are believed to be among the most important targets (6-8). Members of this superfamily include the nicotinic acetylcholine, serotonin type 3, γ-aminobutyric acid type A (GABA A ), and † This work was supported by grants from the National Institutes of Health GM61927 and GM58448 for DER and GM55876 and GM65218 for JSJ LGICs are complex molecules that cannot be expressed in significant quantities and at high purity, there remain considerable obstacles to applying powerful biophysical and biochemical approaches to gain insight into how inhaled anesthetics act on these targets.To overcome these obstacles, small, water-soluble four-α-helix bundle proteins have been designed, produced, and used as well-defined model systems to explore how inhaled anesthetics bind to p...

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“…Isothermal titration calorimetry was performed using a MicroCal VP-ITC titration microcalorimeter (Northampton, MA) at 20 8C (except for in the case of desflurane, where the data were collected at 10 8C) as described [5,6].…”

Section: Methodsmentioning

confidence: 99%

“…These methods include fluorine-19 nuclear magnetic resonance spectroscopy [1], photoaffinity labeling with halothane [2], intrinsic tryptophan fluorescence quenching 0531 [3], and isothermal titration calorimetry [4][5][6]. Although volatile general anesthetic binding affinities for protein targets, or dissociation constants, can be determined with all these techniques, the latter method has the distinct advantage over the other three approaches of allowing both the enthalpic (DH8) and entropic (DS8) contributions to binding to be resolved during a single experiment [7].…”

Section: Introductionmentioning

confidence: 99%

The binding of 10 general anesthetics to a four-alpha-helix bundle protein displays enthalpy–entropy compensation

Zhang

Johansson

2005

International Congress Series

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A calorimetric study on the binding of six general anesthetics to the hydrophobic core of a model protein

Cited by 8 publications

References 25 publications

Expression and Characterization of a Four-α-Helix Bundle Protein That Binds the Volatile General Anesthetic Halothane

Expression and Characterization of a Four-α-Helix Bundle Protein That Binds the Volatile General Anesthetic Halothane

Kinetics of Anesthetic-Induced Conformational Transitions in a Four-α-Helix Bundle Protein

The binding of 10 general anesthetics to a four-alpha-helix bundle protein displays enthalpy–entropy compensation

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