Probing the orientation of reconstituted maltoporin channels at the single-protein level.
Recently we have shown that maltoporin channels reconstituted into black lipid membranes have pronounced asymmetric properties in both ion conduction and sugar binding. This asymmetry revealed also that maltoporin insertion is directional. However, the orientation in the lipid bilayer remained an open question. To elucidate the orientation, we performed point mutations at each side of the channel and analyzed the ion current fluctuation caused by an asymmetric maltohexaose addition. In a second series we used a chemically modified maltohexaose sugar molecule with inhibited entry possibility from the periplasmic side. In contrast to the natural outer cell wall of bacteria, we found that the maltoporin inserts in artificial lipid bilayer in such a way that the long extracellular loops are exposed to the same side of the membrane than protein addition. Based on this orientation, the directional properties of sugar binding were correlated to physiological conditions. We found that nature has optimized maltoporin channels by lowering the activation barriers at each extremity of the pore to trap sugar molecules from the external medium and eject them most efficiently to the periplasmic side.
Danelon
Christophe
C
Institut de Pharmacologie et de Biologie Structurale, Université Paul Sabatier, 31077 Toulouse, France. danelon@ipbs.fr
Brando
Thérèse
T
Winterhalter
Mathias
M
D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005936:Glucans / Q000494:pharmacology; D015640:Ion Channel Gating; D007700:Kinetics; D008051:Lipid Bilayers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018272:Porins / Q000737:chemistry / Q000502:physiology; D011487:Protein Conformation; D011991:Receptors, Virus / Q000737:chemistry* / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D005936:Glucans; D008051:Lipid Bilayers; D018272:Porins; D011991:Receptors, Virus; D011994:Recombinant Proteins; C016532:maltoporins; C031146:maltoheptaose