Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore.
Here we describe a new type of biosensor element for detecting proteins in solution at nanomolar concentrations. We tethered a 3.4 kDa polyethylene glycol chain at a defined site within the lumen of the transmembrane protein pore formed by staphylococcal alpha-hemolysin. The free end of the polymer was covalently attached to a biotin molecule. On incorporation of the modified pore into a lipid bilayer, the biotinyl group moves from one side of the membrane to the other, and is detected by reversible capture with a mutant streptavidin. The capture events are observed as changes in ionic current passing through single pores in planar bilayers. Accordingly, the modified pore allows detection of a protein analyte at the single-molecule level, facilitating both quantification and identification through a distinctive current signature. The approach has higher time resolution compared with other kinetic measurements, such as those obtained by surface plasmon resonance.
Movileanu
L
L
Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.
Howorka
S
S
Braha
O
O
Bayley
H
H
D000911:Antibodies, Monoclonal / Q000276:immunology; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000276:immunology / Q000378:metabolism; D004553:Electric Conductivity; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011506:Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D012996:Solutions; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D007473:Ion Channels; D008024:Ligands; D008051:Lipid Bilayers; D011506:Proteins; D012996:Solutions; C021091:staphylococcal alpha-toxin; D011092:Polyethylene Glycols; D001710:Biotin; D019809:Streptavidin