INTELLECTUAL MERIT: Advances in rational membrane protein design, molecular recognition, and single-molecule technology will be employed to enable chemical sampling at high temporal and spatial resolution, as well as the detection, exploration, and characterization of individual proteins. Partitioning of a protein analyte into a nanopore produces a transient current blockade, the nature of which depends on several factors that will be well-controlled by single-molecule experimental design. Engineered beta-barrel protein pores will be used in single-molecule stochastic sensing, because these systems exhibit a remarkable array of advantageous characteristics, including robustness, versatility and tractability. These studies will be aimed at developing protein nanopore-based biosensors that feature a wider pore diameter to accommodate bulky biopolymers, such as folded proteins and their complexes with interacting ligands. The pore protein will be ferric hydroxamate uptake component A (FhuA), a monomeric beta- barrel protein found in the outer membranes of Gram-negative bacteria. Single-molecule stochastic sensing of the well-studied ribonuclease barnase (Ba), fused to the positively charged leading presequence of the N-terminal of precytochrome b2 (pb2), will be examined in detail. The expected immediate outcomes will be the following: (1) molecular engineering of the FhuA-derived protein nanopore with single and multiple deletions of extracellular loops; (2) unusual stabilization of engineered FhuA-derived nanopores by placing critical covalent and noncovalent intra-molecular contacts at strategic positions within the interior of the nanopore; (3) optimization of the single-molecule experimental design for maximizing the signal-to-noise ratio, thereby quieting the single-channel electrical trace recorded with an engineered FhuA-derived nanopore; (4) single-molecule stochastic sensing of folded proteins and their complexes with interacting ligands; (5) improvement of the detection capabilities of the nanopore-based devices for proteins by engineering internal electrostatic traps at well-designed positions within the interior of the nanopore. Adaptation of these approaches to a microfabricated chip platform will not only provide a new generation of research tools in nanomedicine for examining the details of complex recognition events in a quantitative manner, but will also represent a crucial step in designing nanopore-based biosensors and high-throughput devices for biomedical molecular diagnosis and environmental monitoring.
BROADER IMPACTS: Recent work in the PI's laboratory has demonstrated the capability of nanopore-based biosensors to detect a broad range of analytes, including small organic molecules, polypeptides, polyelectrolytes, neutral polymers, binding proteins and nucleic acids. Studies that focus on engineered nanopores will be rich in information and contribute to fundamental science. Moreover, it is expected that the engineered protein nanopores will have a broad, long-term impact in several arenas, such as biosensing technology, separation-based science, and high-throughput pharmaceutical screening. The proposed experimental plan will be pursued by an integrated approach through strategic partnerships with structural biologists, biophysicists and theoretical chemists. Furthermore, this proposal charts major directions for fostering interdisciplinary research initiatives in Biomaterials and their integration with educational, training and teaching activities at Syracuse University. These will include the following: (1) expanding and diversifying the research opportunities for highly talented undergraduates with a strong desire for a career in science; (2) bolstering the recruitment of undergraduate and graduate science majors from underrepresented groups; (3) adapting the curriculum for the interdepartmental graduate programs in Structural Biology, Biochemistry and Biophysics as well as in Biomaterials, which is in accord with the implementations recommended by the US National Academies.
Intellectual merit. It is well known that proteins are not chemically, mechanically and thermally stable under various experimental contexts departing away from physiological conditions. On the other hand, proteins feature well-defined specific interactions with other biomolecular partners, a characteristic that is not encountered in the inorganic world. Thanks to this award, people in the Movileanu laboratory have been able to redesign and create highly robust protein nanopore scaffolds using membrane proteins existent in nature. This has been accomplished by employing a coupling between genetic engineering and customized protein refolding approaches. Then, such redesigned scaffolds were equipped with functional groups, so that these engineered proteins can transduce signals produced by the presence of various analyte molecules in a given sample. These outcomes represent a critical step towards developing protein-based biosensors for rapid assays in environmental monitoring, molecular biomedical diagnosis, and national security. New biosensors have been created through a combination of different approaches, including rational membrane protein design, molecular recognition and single-molecule technology. Broader Impacts. There is a pressing societal need for the development of devices employed in highly selective screening of a broad range of chemical and biological agents, such as small organic molecules, pollutants, proteins, nucleic acids, viruses, and bacteria. Target areas for such devices not only include warfare chemical and biological detection, but also drug and biomarker discovery as well as genomic and proteomic profiling. Engineered protein-based nanopores resulted from this work will have a profound, long-term impact in various applied arenas, such as biosensor technology, separation-based science and high-throughput pharmaceutical screening. Because of their unusual stability, the newly redesigned protein-based sensors represent an essential bionanostructure platform for further development as well as integration of protein-based components with solid-state devices. This research project has enabled strategic partnerships with structural biologists, biophysicists and theoretical chemists. Through this award, multiple research opportunities for talented undergraduate and graduate students, who have a strong desire for a career in science, have been possible in the area of engineered bionanomaterials. Research internship opportunities for undergraduate students majoring in Bioengineering have catalyzed our local collaborations with Syracuse Biomaterials Institute, a strategic investment in fundamental and applied biomaterials research at Syracuse University. In addition, this laboratory maintained a persistent interaction with science teachers and high-school students from Syracuse district areas, who expressed interest in research exposures at the interface of physics, protein engineering, and biotechnology.
