INTELLECTUAL MERIT: The objective of this proposal is to develop protein springs for use in nanotechnology applications. The proposal has two goals. The first is to characterize how the amino acid sequence of ankyrin (ANK)-repeats controls the stability, shape, spring constant, and elastic limit of ANK repeat proteins. The second goal is to generate shear responsive devices using ANK-repeat protein springs. Two devices will be made. The first will image shear in solution (fluid shear) via Foerster resonance energy transfer (FRET) signals between fluorophors covalently bound to either end of the protein springs. The second device will release a peptide cargo in response to specific shear thresholds. Proteins will be produced in bacteria using expression vectors that encode designed protein sequences. Designed proteins will be characterized for (i) protein stability using circular dichroism spectroscopy in conjunction with thermal and chemical denaturants, (ii) protein shape using cryo-electron microscopy and single particle reconstruction, and (iii) spring constant and elastic limit using atomic force microscopy (AFM). The shear sensor will consist of a single coil that places the N-terminal and C-terminal repeats close in space. The first and last repeats have the only lysine and the only cysteine, respectively, in the protein, allowing simple chemistry to covalently attach different fluorescent dyes to either end. A dye that emits at energies close to the absorption of a second dye can excite the second dye in a distance dependent process referred to as FRET. Shear acts as an extension force that increases the distance between the ends of the protein spring, thereby changing the FRET response. The sensor device will be used to visualize fluid shear in flow cells mounted on a two-photon fluorescence microscope. The shear delivery devices will consist of ANK-repeat proteins in which six of the repeats are designed to form a highly acidic patch that can bind the polypeptide, R9. R9 consists of nine consecutive arginines and has the ability to cross cellular membranes. The delivery device is designed such that at specific extension loads, the R9-binding repeats unfold, disrupting the acidic patch and releasing R9. Release of R9 will be followed using two approaches. The first approach will couple FRET donors to the ANK-repeat protein and a FRET acceptor to R9 and measure FRET as a function of fluid shear in a flow cell attached to a two photon microscope. The second approach will follow the uptake of fluorescent R9 into cells attached to the walls of the flow cell as a function of fluid shear.
BROADER IMPACTS: The proposal describes the design and construction of tunable molecular springs that could be used not only as measures of local shear at the nanoscale but also for the fabrication of active components of many nanodevices. Graduate students will be recruited to this project at both UT Southwestern and at Duke University from their respective graduate student pools. These students will participate at all levels of the project including, but not restricted to, protein expression, protein purification, thermal and chemical denaturation experiments, CD spectroscopy, molecular imaging, single particle analysis, and AFM experiments. It is expected that students will play key roles in experimental design and manuscript preparation in addition to their role in the conduct of experiments. Undergraduate students will participate as summer interns either ad hoc or through the SURF (Summer Undergraduate Research Fellowship) program, which facilitates summer research for undergraduate students at UT Southwestern. The PI will participate in the STARS (Science Teacher Access to Resources at Southwestern) program, which coordinates summer research for high school science teachers at UT Southwestern Medical Center. The STARS program also provides a lecture series for high school science teachers throughout the school year. This lecture series provides both seminars on current research conducted at UT Southwestern and training for research "toolboxes" that are provided to high school teachers for the purpose of illustrating science principals to their high school students. Finally, high school students will be employed either ad hoc or in conjunction with the STARS program, which coordinates a high school summer internship program for high school juniors from the Dallas Independent School District (DISD). The DISD is an urban school district with a high minority demographic and efforts through the STARS program are expected to promote science education of minorities, particularly Hispanic and African American students.
Nanotechnology uses materials that are designed at the molecular level to produce devices on the nanometer scale (nanoscale). Springs are key components of devices; however, the generation of nanoscale springs for nanodevices is currently stymied by an inability to manufacture nanosprings with high precision and poor ability to introduce functionality that will allow nanosprings to be incorporated into larger devices. This proposal addressed both issues using designed protein springs. Protein springs have the potential to solve these issues because proteins are self-forming structures and thus the shape and the spring constant are a function of the amino acid sequence. Control of the amino acid composition also allows functional groups to be coupled to protein springs through protein interactions and directed chemical modifications. The difficulty with designed proteins is the relationships between amino acid sequence and protein function are not fully understood. This proposal used a repeating amino acid sequence as a building block to generate long curved structures that resemble metal springs (Image 1). The first two and half years of the proposal involved iterations of design, synthesis and testing. These iterations led to a final design that is both highly stable and fully soluble under physiological conditions. This final design was then functionalized through the use of specific amino acids to generate a simple sensor device (Image 2). The design of this device allows different fluorescent dyes to be covalently bound to either end of the spring. Proper choice in dyes allows a signal called FRET to be detected as a function of distance between the dyes. Springs can be compressed or extended as a function of force. Our data show that our spring design generates a strong FRET signal and current efforts are testing this device for hydrodynamic flow applications.
