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.
Pidikiti
Ravindernath
R
Departments of Anesthesia, and the Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Shamim
Mohammad
M
Mallela
Krishna M G
KM
Reddy
Konda S
KS
Johansson
Jonas S
JS
D000595:Amino Acid Sequence / Q000235:genetics; D018685:Anesthetics, Inhalation / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D006221:Halothane / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary / Q000235:genetics / Q000502:physiology; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D018685:Anesthetics, Inhalation; D011506:Proteins; D006221:Halothane