title: Design and biophysical characterization of a monomeric four-alpha-helix bundle protein Aα₄ with affinity for the volatile anesthetic halothane.
abstract: A monomeric four-α-helix bundle protein Aα₄ was designed as a step towards investigating the interaction of volatile general anesthetics with their putative membrane protein targets. The alpha helices, connected by glycine loops, have the sequence A, B, B', A'. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and A', B and B', respectively) as different as possible, while using codons which are favorable for expression in E. coli. The protein was bacterially expressed and purified to homogeneity using reversed-phase HPLC. Protein identity was verified using MALDI-TOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein Aα₄. Guanidinium chloride induced denaturation showed that the monomeric four-α-helix bundle protein Aα₄ is considerably more stable compared to the dimeric di-α-helical protein (Aα₂-L38M)₂. The sigmoidal character of the unfolding reaction is conserved while the sharpness of the transition is increased 1.8-fold. The monomeric four-α-helix bundle protein Aα₄ bound halothane with a dissociation constant (K(d)) of 0.93 ± 0.02mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four-α-helix bundle protein can now be used as a scaffold to incorporate natural central nervous system membrane protein sequences in order to examine general anesthetic interactions with putative targets in detail.
authors: University of Pennsylvania School of Medicine, Department of Anesthesiology and Critical Care, 3620 Hamilton Walk, Philadelphia, PA 19104, USA. Lucia.morstadt@gmail.com
authors: Lucia
authors: L
authors: Morstadt
authors: Qing Cheng
authors: QC
authors: Meng
authors: Jonas S
authors: JS
authors: Johansson
mesh_terms: D000595:Amino Acid Sequence; D018685:Anesthetics, Inhalation / Q000378:metabolism*; D055592:Biophysical Phenomena; D002850:Chromatography, Gel; D006221:Halothane / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000378:metabolism*; D012996:Solutions
chemical_list: D018685:Anesthetics, Inhalation; D011506:Proteins; D012996:Solutions; D006221:Halothane