Biological activity and dynamic structure in artificial protein hydrogels

• Main Author: Kennedy, Scott Brian
• Language: ENG
• Published: ScholarWorks@UMass Amherst 2002
• Subjects: Polymers|Chemical engineering
• Online Access: https://scholarworks.umass.edu/dissertations/AAI3056248

Techniques of genetic engineering have recently allowed for the synthesis of artificial, multidomain proteins. One exciting example of such a system is the recently published work of Petka et al describing an artificial protein comprised of three domains. One domain is an artificial sequence of amino acids designed to be highly charged, water soluble, and absent of secondary or tertiary structure. Flanking this domain are two domains designed after the amino acid sequential motif of leucine zippers. Leucine zippers are a naturally occurring class of proteins or protein domains that form α-helical structures and reversibly self-associate in aqueous solution with changes in pH and temperature. This artificial “triblock” protein design behaves as a physical gel in concentrated solutions near physiological conditions. In keeping with the exciting promise for future development of this system, three projects develop. First, incorporation of a site susceptible to hydrolysis by thrombin demonstrates a biologically active hydrogel. As site specific activity was not present in Petka's original design, new protein designs incorporate an enzyme cleavable site at both ends of the water-soluble coil domain. Gels prepared from the new protein irreversibly liquefy upon exposure to thrombin, and a complete kinetic analysis indicates successful incorporation of site-specific biological activity. Second, ultracentrifugation and small angle x-ray scattering (SAXS) were used to elucidate the gel structure. Dilute solution ultracentrifugation indicates tetrameric association of the leucine zipper domain. SAXS profiles from gel samples are also consistent with a large population of tetrameric aggregates. Together these results strengthen the hypothesis that leucine zipper associations act as physical crosslinks. Finally, the evidence for a well-defined and controllable gel structure provided by SAXS led to a study of gel dynamics by diffusing wave spectroscopy (DWS). Coupled with fluorescence resonance energy transfer experiments, DWS results indicate that macroscopic physical properties correlate with molecular dynamics. This result opens the door to a wide range of potential and promising applications for precision-engineered physical protein hydrogels.