INTELLECTUAL MERIT: The project will combine membrane protein design with high resolution single-channel electrical recording and adapt these to the lab-on-a-chip format. This strategy permits design of powerful stochastic sensing elements for the detection of double-stranded DNA (dsDNS) and proteins. A prerequisite for a stable stochastic sensing element is a robust protein channel scaffold. Beta-barrel membrane proteins fulfill this requirement. The network of hydrogen bonds confers extraordinary rigidity to the core of the beta-barrel, and this attribute has made them open to remodeling in various ways. The fundamental advantage of a protein channel-based sensing element is that it need not be highly selective, because each translocating biomolecule will produce a distinctive electrical signal. This feature reduces the demands on protein engineering, which is otherwise required for detection of small analytes.
The multimeric character of many of beta-barrel proteins makes them less than ideal for remodeling work. This project will employ ferric hydroxamate uptake component A (FhuA), a monomeric beta-barrel protein that is a member of the bacterial outer membrane protein superfamily with a pore size large enough to accommodate both dsDNA and proteins. The high resolution X-ray crystal structure of FhuA has confirmed its monomeric character and has elucidated the channel architecture rather clearly at the atomic level, thereby paving the way for use of this outer membrane protein in channel redesign studies. The crystal structure furthermore reveals a globular N-terminal domain dubbed the "cork" that would appear to impede transit through the channel but that is instead vital to its function. This feature invites investigation via recombinant protein engineering of the physical basis for large molecule transport. The monomeric character of FhuA renders genetic manipulation of the protein more straightforward. The expected major outcome of these studies will be a comprehensive picture of the stochastic biosensor based on wild-type and redesigned channels. This will lay the foundation for further remodeling work on beta-barrel channel proteins. The proposed experiments will be carried out in collaboration with German partners who will provide wild-type and mutant FhuA, other German partners who will help with implementation at the lab-on-a-chip level, and with US computational physicists who will conduct parallel theoretical studies of membrane transport dynamics using molecular dynamics simulations.
BROADER IMPACTS: This study will move the field of membrane transport channels forward, especially because FhuA has the physical dimensions to accommodate transport of larger biomolecules than can be transported by the better known alpha-hemolysin. The work provides entry to study of the transport of DNA and large proteins across cell boundaries with very important implications for understanding this process and for potential therapeutic uses. The proposed lab-on-a-chip device has important potential applications in separation science, drug delivery, biosensing, and gene and biologics therapies. The proposal has a well developed approach to mentoring undergraduate research students, providing both an expectation of substantial student commitment of time and effort and significant rewards for productive students in terms of possible exchange study in the lab of one of the German collaborators. The PI is also engaged in upgrading the interdisciplinary Biophysical Sciences program at Syracuse, including the introduction of a new course in biosensors.
