Design and biophysical characterization of a monomeric four-alpha-helix bundle protein A alpha 4 with affinity for the volatile anesthetic halothane

A monomeric four- alpha -helix bundle protein A alpha 4 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, Ba2, Aa2. The DNA sequence was designed to make the helices with the same amino acid sequences (helix A and Aa2, B and Ba2, 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 MALDIaTOF mass spectrometry. Far-UV circular dichroism spectroscopy confirmed the predominantly alpha-helical nature of the protein A alpha 4. Guanidinium chloride induced denaturation showed that the monomeric four- alpha -helix bundle protein A alpha 4 is considerably more stable compared to the dimeric di- alpha -helical protein (A alpha 2-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- alpha -helix bundle protein A alpha 4 bound halothane with a dissociation constant (Kd) of 0.93 +/- 0.02 mM, as shown by both tryptophan fluorescence quenching and isothermal titration calorimetry. This monomeric four- alpha -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.