Single-molecule Stochastic Sensing of DNA and Proteins Liviu Movileanu (Syracuse University)DMR 0706517 Double - stranded DNA Proteins DNA DNA FhuA FhuA bilayer bilayer Folded barnase domain ( Ba ) ?? ?? Lipid bilayer Leading presequence (pb ) 2 Entry trap + V current current time time open open _ t t t t off off on on Exit trap Engineered FhuA Nanopore closed closed Our laboratory is now able to control the interaction of a single protein with a transmembrane protein pore at single-molecule resolution. Electrostatic traps, which consist of negatively charged residues, were engineered at strategic positions within the pore lumen. The electrostatic attraction between the traps and the leading fragment of the protein analyte is the key mechanism by which sensing sensitivity and selectivity is improved substantially. In general, this technique can be applied to single-molecule stochastic sensing of short polypeptides (J. Am. Chem. Soc. 129(45), 14034-14041, 2007) and small folded protein domains (J. Am. Chem. Soc. 130(12), 4081-4088, 2008) using engineered nanopores and resistive-pulse technique. Stochastic sensing of double-stranded DNA and proteins is used to concurrently reveal the rate constants of association and dissociation of the biopolymer to the engineered nanopore. We have expanded the use of single-molecule stochastic sensing to a variety of engineered nanopores, which are related in structure and sequence homology to outer membrane proteins from E.coli. Such a biosensing approach enables the determination of both the identity and concentration of protein or DNA analytes. Transient current blockades shown above represent individual bindings of the protein analyte to the engineered FhuA-based nanopore.
Single-molecule Stochasting Sensing of DNA and Proteins Liviu Movileanu (Syracuse University) DMR 0706517 Education: The research team that contributed to the preliminary stage of this work included several undergraduate researchers (Aaron Woolfe, Carl P. Goodrich, Robert Bikwemu and Tatiana Konyakhina), two graduate students (Khalil Howard and David Niedzwiecki) and one postdoctoral researcher (Mohammad M. Mohammad). Aaron J. Wolfe is a fist author on one of the JACS papers pertinent to this work. Two other undergraduates, Carl Goodrich and Catalin Chimerel (a visitor from Germany) co-author papers in J. Phys. Chem. and Eur. Biophys. J. Impact: There are many exciting prospects for the utilization of engineered protein nanopores in various arenas, including separation-based science, drug delivery, synthetic biology and biotherapeutics. We have shown that eventually these protein nanopores could be used as integral elements of biosensors for single-molecule stochastic sensing of short peptides and folded protein domains. The adaptation of these approaches to a microfabricated chip platform will provide a new generation of research tools for examining the details of complex recognition events in a quantitative manner. This research represents a crucial step in the design of nanopore-based biosensors and high-throughput devices for biomedical molecular diagnosis, environmental monitoring, and homeland security. Undergraduate researcher Carl Goodrich analyzes his single-molecule nanopore measurements that employed beta-hairpin peptides. The voltage-clamp instrument illustrated in the above picture was purchased using funds from the NSF/DMR-706517 award.