Design and Biophysical Characterization of a Single Chain Four-alpha-helix Bundle Protein Which Binds Volatile General Anesthetics

Abstract

In order to investigate the interaction of volatile general anesthetics with their putative membrane protein targets, we designed a four-alpha-helix bundle protein in a single protein chain. The four alpha helices, connected by three 8 glycine loops, had the sequence A, B, B’, A’. The DNA sequence was designed with the goal of making helices with the same amino acid sequence (helix A and A’, B and B’, respectively) as different as possible in their DNA sequence, while using codons which are favorable for expression in E.coli. Restriction enzyme sites were added on both ends. The synthesized DNA sequence was cloned into an expression vector. The protein was bacterially expressed and purified to homogeneity using reverse-phase HPLC. Protein identity was verified using MALDI-TOF mass spectroscopy. Near UV circular dichroism spectroscopy confirmed the strongly alpha helical nature of the protein. Guanidinuium chloride denaturation showed that the single chain four-alpha-helix bundle protein is twice as stable as the dimeric di-helical protein. The sigmoidal character of the unfolding reaction was conserved, the sharpness of the transition increased. Our single chain four-alpha-helix bundle protein bound halothane with a dissociation constant of 1.2 mM, as shown by tryptophan fluorescence quenching. This single chain four-alpha-helix bundle protein can now be used as a scaffold to incorporate natural membrane protein sequences to examine general anesthetic interactions in detail.