Biomolecular Materials: Structure, Phase Behavior, and Interactions
INTELLECTUAL MERIT: The project aims to elucidate the nature of structures and interactions in self-assemblies of filamentous neurofilaments derived from the axonal cytoskeleton and structures of curvature stabilizing lipids. The proposal combines synchrotron small angle- x-ray-scattering (SAXS), optical and cryo-electron electron microscopy experiments with statistical mechanical theoretical investigations. One series of proposed experiments focuses on understanding the nature of interfilament interactions in neurofilament networks reconstituted from purified proteins. Neurofilaments (NFs) consist of three homopolymers NF-L, NF-M, and NF-H. Recent preliminary data from the PI?s laboratory suggest qualitatively different behavior for interfilament interactions with sidearms consisting of either NF-M or NF-H. Using the synchrotron SAXS-osmotic pressure technique, a series of direct force measurements are proposed, which when combined with complementary phase behavior studies, should result in a comprehensive understanding of the nature of the interactions between neurofilaments. The studies will be conducted in NF gel phases as a function of sidearm grafting densities in binary (NF-LH, NF-LM), and ternary (NF-LMH) mixtures. The co-PI?s group will carry out statistical mechanical studies of interactions between polyampholyte brushes mimicking the NF sidearm structure of NF-L, NF-M, and NF-H. Another set of proposed experiments is based on the PI's recent discovery of block liposomes resulting from membrane curvature stabilizing multivalent lipids. Cryogenic-TEM revealed the blocks to consist of distinctly shaped nanoscale spheres, pears, tubes, or rods. Indeed, similar membrane shape changes, occurring in vivo for the purpose of specific cellular functions, are often induced by interactions between membranes and curvature stabilizing proteins. By employing a series of lipids custom synthesized in the PI?s group, which allow for systematic variations in the shape, size and charge of the curvature stabilizing lipids (mimicking properties of more complex curvature stabilizing proteins), the proposed experiments will permit the PI to distinguish between the separate contributions of charge and lipid shape responsible for the formation of block liposomes.
BROADER IMPACTS: From a broader perspective, aside from further enhancing our understanding of polyelectrolyte brushes (which remains poorly understood, both in experiment and theory), the research should enhance the understanding of structures and interactions of the axonal cytoskeleton. The nanorods and nanotubes observed in the studies of block liposomes have potential applications in the area of templating (e.g. to produce nanowires) or chemical delivery. The proposed biomolecular materials program is multidisciplinary and obligates the PIs to educate and train undergraduate and graduate students, and postdoctoral researchers in modern methodologies required to address important problems at the interface between physics, chemistry, engineering, and biology. The state-of-the-art characterization capabilities developed in the PI?s group are integrated within the UCSB Materials Research Science and Engineering Center (MRSEC) x-ray facility and open to a very wide user base comprised of over 30 groups from across campus. Students also gain familiarity with existing national research facilities. The PI and co-PI actively participate in UCSB Outreach Programs with the community colleges and colleges and universities outside of Santa Barbara, which encourage participation by under-represented groups. These programs include, the MRSEC outreach program, the Internships in Nanosystems Science and Engineering Technology (INSET) outreach program, and the Research Experience for Teachers (RET) outreach program. These programs allow the PIs to train and educate undergraduate students and high school teachers in multidisciplinary methods of science and engineering.
The project outcomes relating to the intellectual merit component of the award were related to studies aimed at discovering and understanding phase behavior in certain important lipid systems and proteins that tend to make filamentous structures in cells enabling their biological functions, for example, in the transport of molecules between different locations in cells. The project’s unique approach combined state-of-the-art synchrotron x-ray scattering methods at the National Facility at the Stanford Synchrotron Radiation Laboratory in parallel with theoretical modeling to enable a better understanding of the underlying science. One series of studies leading to an important project outcome was on the discovery of a method to produce a novel gyroid lipid phase, which incorporates functional small RNA molecules for gene silencing. The complex structure of the Gyroid phase consisting of two intertwined but distinct water domains was solved by synchrotron x-ray scattering (to visualize a gyroid phase see e.g. http://en.wikipedia.org/wiki/Gyroid). The significance of this project outcome is due to the remarkable "pore forming" property of gyroid phases resulting from their unusual membrane shapes. The experimental results showed that the gyroid lipid phase-RNA complex was able to interact with biological cells and penetrate key membrane barriers to deliver small RNA molecules for gene silencing. Another important project outcome was the development of a micro-fluidic based method to produce aligned filamentous protein networks for imaging and phase behavior studies in a geometry, which mimics the confined conditions inside biological cells. The resulting network structure was probed by polarized optical microscopy and atomic force microscopy, which confirmed an unprecedented degree of protein alignment within microchannels. This technique is expected to enable structural studies of a range of biological polymers (e.g. based on proteins and nucleic acids DNA or RNA) paving the way for the understanding of the phase behavior under biologically relevant geometrically confined conditions. The project outcome, from a broader impact perspective, has led to the graduation of successful graduate students and postdoctoral researchers, equipped in addressing important problems, at the interface between physics, chemistry, engineering and biology, for careers in academe, national laboratories, and industry. The principal investigators actively participated in UC-Santa Barbara Outreach Programs with the community colleges, and colleges and universities outside of Santa Barbara, which encourage participation by under-represented groups. A very significant project outcome of this award included the training of undergraduates and high school teachers in the following internship programs: the Internships in Nanosystems Science and Engineering Technology (INSET, 1 undergraduate student, summer 2011); Research Internship in Science and Engineering (RISE, 2 undergraduate students in 2008 and 2009); California Alliance for Minority Participation, which promotes diversity of undergraduates in research (CAMP, 3 undergraduate students in 2008, 2010, and 2011); Cooperative International Science and Engineering Internships (CISEI, 1 in 2009); and the Research Experience for Teachers Program (RET, 1 teacher in 2009 and 2 teachers in 2010).
