INTELLECTUAL MERIT: The nuclear pore complex (NPC) mediates transport of materials between the nucleus and cytoplasm of eukaryotic organisms. Current research indicates this unique transport mechanism relies on a protein-based hydrogel, which is both highly selective and capable of operating against concentration gradients. A well-resolved structural model of the NPC has been recently determined. This model and related reports have elucidated many aspects related to the mechanism of transport through the NPC, however critical features of this selective hydrogel phase are not understood. The long term goal of the proposal is to understand the fundamental mechanisms that govern the operation of this protein-hydrogel. The PI would also like to translate this knowledge to synthetic-based biomaterials capable of performing selective separations and sensing in ex-vivo platforms. The central hypothesis of this proposal is that by expressing selected proteins found in the NPC and rationally mutating the structure of these expressed proteins, a better picture of the molecular mechanisms of transport through the NPC can be developed. Further, it is hypothesized that this enhanced understanding will allow development of model semi-synthetic polypeptide hydrogels that will display biomimetic transport properties. This hypothesis has been formulated based on literature reports and preliminary studies, which show proteins from the NPC can be coerced to form selective hydrogels in ex-vivo environments. Specifically, the PI will: (1) determine the relative interactions and properties of proteins located within the nuclear pore complex that are necessary to reproduce selective transport observed in the nuclear pore complex, (2) determine the influence of the hydrophilic regions of the protein hydrogel on transport and the extent to which these regions can be used to synthetically alter transport properties, and (3) develop a route for generating a semi-synthetic hydrogel using solid phase peptide synthesis.
BROADER IMPACTS: Biological systems offer remarkable insight and inspiration for rational materials design. To advance current capabilities, fundamental advances in how biological mechanisms, such as nucleocytoplasmic transport, operate must be realized. The themes upon which this research is based ? biomaterials and bio/analytical chemistry ? can be used to engage students, especially if the subject matter is approached in an appropriate manner. To this end, the PI has initiated an educational outreach program that is integrated with the subject matter of the research program and involves the participation of graduate students in the group. The outreach program uses visual learning concepts and information dissemination using video conferencing in coordination with podcasting technology, forming specific modules that consist of a podcast, a classroom exercise, a follow-up live video conference, and a short assessment of the exercise. Initially, they are focusing on collaboration with two high schools, one of which serves predominantly underrepresented groups. Both high schools are located hundreds of miles (1300 and 800, respectively) from the PI?s laboratory. This is intended to prove that the educational program under development can use the internet effectively to bridge both geographic and demographic divides. To increase the impact of the program the PI makes initial personal visits to the collaborating schools and participates in interactive question-and answer sessions throughout the semester using video conferencing. Along with this collaboration, additional content will be developed and disseminated in separate podcasts that specifically target chemistry students at the high school, undergraduate, and graduate levels. Work is also disseminated through peer-reviewed journals. Pedagogical assessment of the program will be performed in collaboration with Indiana University campus facilities.
In this project, we have developed a synthetic mimic of a gel that controls the movement of molecules between the nucleus and cytoplasm of a cell. The biological gel, found in the nuclear pore complex, displays unique selectivity in how molecules are moved across the nuclear membrane. This selectivity operates on poorly understood principles, but is highly conserved across eukaryotic organisms, and as such is expected to be a very efficient and robust transport mechanism. We have developed a hydrogel comprised of synthetic polymers grafted with small peptide motifs that can mimic some of the selective transport properties of the nuclear pore complex. By tuning the polymeric components (e.g. monomers used), selectivity in charge and hydrophobicity were examined. Further, the effect of grafting density of peptides on transport was examined. These studies open new avenues of inquiry and provide a model system for the study of new biological transport mechanisms. Results of this project have been disseminated in journal publications and through conference presentations. Through the course of this project, graduate students were trained in bioanalytical chemistry and molecular biology protocols. Students were also trained to develop analytical problem solving skills. Outreach events to elementary schools and Indiana Boy Scout Troops were performed during the course of this project as well.
