A genetically encoded pore for the stochastic detection of a protein kinase.
Stochastic sensing is an emerging approach for the detection of a wide variety of analytes at the level of individual molecules. Detection is accomplished by observing the modulation of the current that flows through a single protein pore that has been engineered to bind an analyte of interest. Previously, protein analytes have been detected by using pores to which ligands have been appended at specific sites by targeted chemical modification. Here, we report the first genetically encoded stochastic sensor element for detecting a protein. A protein kinase inhibitor peptide sequence was incorporated into the alpha-hemolysin polypeptide, which was used to form a heteroheptameric pore containing a single copy of the inhibitor sequence. With this pore, the successful detection of the catalytic subunit of protein kinase A was demonstrated. This development should greatly facilitate the detection of active kinase subunits by stochastic sensing and the rapid screening of kinase inhibitors by an approach that yields kinetic information.
Cheley
Stephen
S
Department of Molecular and Cellular Medicine, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.
Xie
Hongzhi
H
Bayley
Hagan
H
D001483:Base Sequence; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D005818:Genetic Engineering; D006460:Hemolysin Proteins / Q000235:genetics*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013269:Stochastic Processes
D004791:Enzyme Inhibitors; D006460:Hemolysin Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C518603:protein kinase inhibitor peptide (5-24); D017868:Cyclic AMP-Dependent Protein Kinases