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

Abstract

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.

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

“…Fast and slow components of quenching occur as an anesthetic shifts the conformational equilibrium towards the higher affinity states because the W15 quantum yield in these states is lower. Consistent with this model, the K d for halothane determined from the immediate component of quenching is six-fold higher than that reported at equilibrium (0.68 ± 0.08 mM vs. 0.12 ± 0.02 mM, respectively) (23). Application of this model to interpret the actions of anesthetics on Cys-loop LGICs leads to the prediction that anesthetics bind with higher affinity to the open channel state as compared to the closed channel state.…”

Alzheimer’s Disease: Halothane Induces Aβ Peptide to Oligomeric Form—Solution NMR Studies

“…It has been shown that bound volatile general anesthetics alter both local protein dynamics and global protein stability [65][66][67][68]. Both Ab40 and Ab42 peptides have two a-helices (a-helix-I (residues 15-23), a-helix-II (residues 32-36)) connected by a more flexible kink region.…”

Binding of small molecules to cavity forming mutants of a de novo designed protein

“…Such sites can be engineered into pre-existing natural protein scaffolds, [1][2][3][4][5] or into proteins designed de novo. [6][7][8][9][10][11][12][13][14] In general, two approaches are used to produce novel binding proteins: (i) In some cases, researchers use principles of rational design to engineer hydrophobic packing and buried polar interactions in the core of a protein to facilitate binding of a small molecule. [6][7][8]15 (ii) Alternatively, high throughput methods, such as phage display or mRNA display, can be used to find rare binding proteins amidst large combinatorial libraries of nonbinding sequences.…”