Binding of the General Anesthetics Chloroform and 2,2,2-Trichloroethanol to the Hydrophobic Core of a Four–α-Helix Bundle Protein¶

Abstract

The structural features of general anesthetic binding sites on proteins are being examined using a defined model system consisting of a four-alpha-helix bundle scaffold with a hydrophobic core. Previous work suggested that halothane binding to the four-alpha-helix bundle was improved by (1) introducing a cavity into the hydrophobic core and (2) substituting a methionine side-chain in place of an alpha-helical heptad e position leucine. In this study, the ability of the general anesthetics chloroform and 2,2,2-trichloroethanol to bind to the hydrophobic core of the four-alpha-helix bundle (Aalpha2-L38M)2 is explored. The halogenated alkane chloroform binds with a dissociation constant (Kd) = 1.4 +/- 0.2 mM, whereas 2,2,2-trichloroethanol binds with a Kd = 19.5 +/- 1.2 mM. The affinity of both general anesthetics for the hydrophobic core of the four-alpha-helix bundle approximates their whole animal effective concentration in 50% of test subjects' (EC50) values, as shown previously for halothane. Tryptophan phosphorescence decay rates at 77 K are accelerated by a factor of 4.5 by both bound halothane and chloroform, indicating that the heavy-atom effect is responsible for a portion of the observed fluorescence quenching. Because heavy-atom effects are operative only at short distances, the findings indicate that these general anesthetics are binding in the vicinity of the indole rings of W15 in the hydrophobic core of the four-alpha-helix bundle scaffold. The results indicate that chloroform, halothane and 2,2,2-trichloroethanol may occupy the same sites on protein targets.

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

“…2A), and the DH8, K d , the free energy change associated with binding (DG8) and the change in entropy associated with binding (DS8) were determined from the plots. The K d value of 880F10 AM is quite comparable to the value of 1.4F0.2 mM obtained using tryptophan fluorescence quenching [14] supporting the validity of the results. Table 1 contains the remaining thermodynamic parameters underlying chloroform binding to the four-a-helix bundle (Aa 2 -L38M) 2 .…”

“…The anesthetic EC 50 values for desflurane and fluroxene in humans are 0.52 and 0.95 mM, respectively [25]. Two data points are for chloroform, using the current K d value and that of 1.4F0.2 mM determined using fluorescence spectroscopy as reported earlier [14]. There are also two data points for halothane, indicating the K d values determined using fluorescence quenching [7] and isothermal titration calorimetry [13].…”

“…Earlier work using tryptophan fluorescence quenching indicates that chloroform binds to the four-a-helix bundle (Aa 2 -L38M) 2 with a K d of 1.4F0.2 mM [14]. To achieve a c value in the ideal range for isothermal titration calorimetry (5-50) would therefore require prohibitively high concentrations of protein (on the order of 7-70 mM).…”

“…The current work builds on an earlier study using isothermal titration calorimetry [13] that characterized the binding of halothane, isoflurane, enflurane and sevoflurane to the same protein. The binding of chloroform to the four-ahelix bundle (Aa 2 -L38M) 2 as determined using isothermal titration calorimetry is first investigated in order to further calibrate the validity of the results against prior fluorescence quenching data examining this interaction [14]. The thermodynamic characterization of the binding of bromoform, trichloroethylene, benzene, desflurane and fluroxene to the four-a-helix bundle (Aa 2 -L38M) 2 is then presented.…”

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

“…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 (17)(18)(19)(20)(21). Each four-α-helix bundle protein is a dimer of two 27-residue α-helices joined by an eight-residue glycine linker (figure 1).…”