Controlling a single protein in a nanopore through electrostatic traps.
Protein-protein pore interaction is a fundamental and ubiquitous process in biology and medical biotechnology. Here, we employed high-resolution time-resolved single-channel electrical recording along with protein engineering to examine a protein-protein pore interaction at single-molecule resolution. The pore was formed by Staphylococcus aureus alpha-hemolysin (alphaHL) protein and contained electrostatic traps formed by rings of seven aspartic acid residues placed at two different positions within the pore lumen. The protein analytes were positively charged presequences (pb2) of varying length fused to the small ribonuclease barnase (Ba). The presence of the electrostatic traps greatly enhanced the interaction of the pb2-Ba protein with the alphaHL protein pore. This study demonstrates the high sensitivity of the nanopore technique to an array of factors that govern the protein-protein pore interaction, including the length of the pb2 presequence, the position of the electrostatic traps within the pore lumen, the ionic strength of the aqueous phase, and the transmembrane potential. Alterations in the functional properties of the pb2-Ba protein and the alphaHL protein pore and systematic changes of the experimental parameters revealed the balance between forces driving the pb2-Ba protein into the pore and forces driving it out.
Mohammad
Mohammad M
MM
Department of Physics, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130, USA.
Prakash
Sumit
S
Matouschek
Andreas
A
Movileanu
Liviu
L
D001224:Aspartic Acid / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering; D012260:Ribonucleases / Q000737:chemistry*; D055672:Static Electricity; D013997:Time Factors
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; C021091:staphylococcal alpha-toxin; D001224:Aspartic Acid; D012260:Ribonucleases