title: Total chemical synthesis of a membrane protein domain analogue containing two transmembrane helices: functional reconstitution of the semisynthetic sensory rhodopsin/transducer complex.
abstract: Negative phototaxis in Archaea is mediated by the sensory rhodopsin II/transducer complex (NpSRII/NpHtrII). After light excitation, the signal is relayed from the receptor to NpHtrII where a rotary motion of TM2 in the membrane domain (NpHtrII1-114) is induced. This conformational change is transferred to the downstream two-component signaling cascade. Here, we describe the chemical synthesis of this membrane domain, which consists of the two transmembrane helices TM1 and TM2. NpHtrII1-114 was synthesized using two sequential ligation steps. The first ligation between NpHtrII47-59 and NpHtrII60-114 was performed in organic solvents, whereas the final ligation was successful in an aqueous buffer that contained a detergent and a denaturant. The product was refolded into micelles and showed functional properties as determined by binding studies to its cognate receptor NpSRII and by photocycle experiments. This work demonstrates that membrane proteins can be successfully synthesized by chemical means paving the way for tailor-made modifications.
authors.affiliation: Max Planck Institute of Molecular Physiology, Otto Hahn Str. 11, 44227, Dortmund, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Dittmann
authors.affiliation: Max Planck Institute of Molecular Physiology, Otto Hahn Str. 11, 44227, Dortmund, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Seidel
authors.affiliation: Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
authors.forename: Igor
authors.initials: I
authors.lastname: Chizhov
authors.affiliation: Max Planck Institute of Molecular Physiology, Otto Hahn Str. 11, 44227, Dortmund, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Engelhard
mesh_terms: D000595:Amino Acid Sequence; D060326:Chemistry Techniques, Synthetic; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D025603:Sensory Rhodopsins / Q000138:chemical synthesis* / Q000737:chemistry
chemical_list: D008565:Membrane Proteins; D025603:Sensory Rhodopsins
keywords: archaeal phototaxis; ligation in organic solvents; membrane proteins; protein folding; protein synthesis