Single-molecule observation of the catalytic subunit of cAMP-dependent protein kinase binding to an inhibitor peptide.
An engineered version of the staphylococcal alpha-hemolysin protein pore, bearing a peptide inhibitor near the entrance to the beta barrel, interacts with the catalytic (C) subunit of cAMP-dependent protein kinase. By monitoring the ionic current through the pore, binding events are detected at the single-molecule level. The kinetic and thermodynamic constants governing the binding interaction and the synergistic effect of MgATP are comparable but not identical to the values in bulk solution. Further, the values are strongly dependent on the applied membrane potential. Additional exploration of these findings may lead to a better understanding of the properties of enzymes at the lipid/water interface. Despite the complications, we suggest that the engineered pore might be used as a sensor element to screen inhibitors that act at either the substrate or ATP binding sites of the C subunit.
Xie
Hongzhi
H
Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, 440 Reynolds Medical Building, College Station, TX 77843-1114, USA.
Braha
Orit
O
Gu
Li-Qun
LQ
Cheley
Stephen
S
Bayley
Hagan
H
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D015374:Biosensing Techniques / Q000379:methods; D002384:Catalysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry; D013816:Thermodynamics; D013997:Time Factors
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D010455:Peptides; D021122:Protein Subunits; C021091:staphylococcal alpha-toxin; D000255:Adenosine Triphosphate; D017868:Cyclic AMP-Dependent Protein Kinases