Stochastic sensing of nanomolar inositol 1,4,5-trisphosphate with an engineered pore.
The introduction of a ring of arginine residues near the constriction in the transmembrane beta barrel of the staphylococcal alpha-hemolysin heptamer yielded a pore that could be almost completely blocked by phosphate anions at pH 7.5. Block did not occur with other oxyanions, including nitrate, sulfate, perchlorate, and citrate. Based on this finding, additional pores were engineered with high affinities for important cell signaling molecules, such as the Ca(2+)-mobilizing second messenger inositol 1,4,5-trisphosphate (IP(3)), that contain phosphate groups. One of these engineered pores, P(RR-2), provides a ring of fourteen arginines that project into the lumen of the transmembrane barrel. Remarkably, P(RR-2) bound IP(3) with low nanomolar affinity while failing to bind another second messenger, adenosine 3', 5'-cyclic monophosphate (cAMP). The engineered alpha-hemolysin pores may be useful as components of stochastic sensors for cell signaling molecules.
Cheley
Stephen
S
Department of Medical Biochemistry and Genetics, The Texas A & M University System Health Science Center, College Station, TX 77843, USA. scheley@medicine.tamu.edu
Gu
Li Qun
LQ
Bayley
Hagan
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D015374:Biosensing Techniques; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D015544:Inositol 1,4,5-Trisphosphate / Q000032:analysis* / Q000378:metabolism; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D036103:Nanotechnology / Q000379:methods; D010710:Phosphates / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D010710:Phosphates; C021091:staphylococcal alpha-toxin; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; D001120:Arginine; D008274:Magnesium