"NIRT: Intein Proteins as Nanoswitches for Biotechnology: Linking Molecular Modeling with Biophysical and Genetic Methods"
This is a four-year cross-disciplinary, multi-investigator, multi-institutional proposal focused on an autocatalytic self-processing protein called an intein, which will be adapted as a nanoswitch. The goals of this research project are to determine the underlying principles of the splicing and cleavage reactions that occur during protein processing and to use this understanding to design a molecular nanoswitch that exhibits desirable properties for use in functional genomics and proteomics. This work will culminate in the use of the nanoswitch to perform protein separation on a fluidics chip platform. The research approach will involve combining classical molecular dynamics and quantum ab initio calculations with biophysical and genetic methods. Together classical and quantum calculations will provide molecular-level insights into the reaction mechanism to help identify amino acids critical to the cleavage reaction and guide the site-directed mutagenesis towards development of smaller, faster cleaving and specifically controllable mutants. Combining this body of knowledge with that from biophysical measurements and performance improvements obtained through mutagenesis, molecular nanoswitches with desirable characteristics for applications in nano-biotechnology will be obtained. The specific aims are to:
1. Use classical molecular dynamics to elucidate the role, the three-dimensional spatial location and the movement of critical amino acids, salt molecules and occluded water molecules in the intein cleavage process during excursions in pH, temperature and other conditions at the splice junctions, and then use these results for quantum ab initio calculations to determine the likely bond cleavage sites as a function of excursions in pH and temperature.
2. Measure intein conformational dynamics during the cleavage reaction using euterium-exchange and time-resolved electrospray ionization-mass spectrometry (ESI-MS), Fluorescence Resonance Energy Transfer (FRET), and measure the secondary structural changes during cleavage using circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy.
3. Use the results from 1 and 2 above, and X-ray crystallographic structures of inteins, to guide specific mutagenesis of inteins to obtain cleavage mutants and cleavage peptides with reduced size, increased cleavage rates, and alternative trigger mechanisms. In addition, combine random mutagenesis with genetic selection schemes and phage display to select for additional derivatives with desirable characteristics.
4. Test newly developed molecular nanoswitches on a fluidics-chip platform for one-step protein recovery for a proteomics application.
Broader Impacts: The proposed approach is novel and uses talents of RPI and Wadsworth Center faculty in a new and synergistic way to address a significant bio- and nano-technology problem. The proposed work is cross-disciplinary (physics, chemical engineering, and genetics/biochemistry) and builds upon previous cross-institutional success developing intein derivatives with useful characteristics for biotechnology. Successful development of controllable nanoswitches will have impact on proteomics (bioseparations on fluidics chip platform) and medicine (biosensors and drug delivery) through collaboration with industry and with other academic labs.
Graduate and undergraduate students will be exposed to interdisciplinary training that spans molecular modeling, biophysical characterization, biochemical engineering and molecular genetics. New curricula for students and web-based visual learning for children has been initiated and will be emphasized in this project. We are also involved in assisting a new program, New Visions, which is interested in attracting high-school students from economically disadvantaged sections of society.
In summary, the proposed research will have a broad impact on advancing discovery and understanding of protein processing while promoting teaching, training and learning in nanoscale science and engineering. The project is supported by CTS/ENG, Biology, and INT NSF organizations.
