A Modular Approach to the Design of Protein-Based Smart Gels Tijana Z. Grove, 1 Jason Forster, 2 Genaro Pimienta, 1 Eric Dufresne, 2,3,4 Lynne Regan 1,5* 1 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511 2 Department of Mechanical Engineering and Materials Science, Yale University, New Haven, CT 06511 3 Department of Physics, Yale University, New Haven, CT 06511 4 Department of Cell Biology, Yale University, New Haven, CT 06511 5 Department of Chemistry, Yale University, New Haven, CT 06511 Received 22 August 2011; revised 4 January 2012; accepted 23 January 2012 Published online 10 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22033 This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com INTRODUCTION H ydrogels with tunable properties and stimuli- responsiveness have great potential for applications in tissue engineering and as therapeutic delivery devices. 1–5 An important emerging theme is the incorporation of noncovalent, specific intermolecu- lar interactions to control assembly and disassembly of bio- molecule-based hydrogels. 4 A variety of different approaches have been suggested including the incorporation of noncova- lent crosslinks based on peptide folding, 6–8 peptide-polysac- charide, 9 and protein–peptide interactions. 10,11 The goal still remains, however, to rationally specify the macroscopic properties of a material by appropriately designing the constituent molecules and microscopic interac- tions. Natural proteins exhibit a breathtaking range of inter- action specificities and affinities, and the intrinsic reversibil- ity of such noncovalent interactions provides a direct route to stimuli-responsive crosslinks in a material. 12,13 All the in- formation required to specify the folded, functional form of a protein is contained in its amino acid sequence. Current A Modular Approach to the Design of Protein-Based Smart Gels *Present address: Department of Chemistry, Virginia Tech, Blacksburg, 24061. Contract grant sponsor: NSF CAREER Contract grant number: CBET-0547294 Correspondence to: Lynne Regan; e-mail: lynne.regan@yale.edu ABSTRACT: The modular nature of repeat proteins makes them a versatile platform for the design of smart materials with predetermined properties. Here, we present a general strategy for combining protein modules with specified stability and function into arrays for the assembly of stimuli-responsive gels. We have designed tetratricopeptide repeat (TPR) arrays which contain peptide-binding modules that specify the strength and reversibility of network crosslinking in combination with spacer modules that specify crosslinking geometry and overall stability of the array. By combining such arrays with multivalent peptide ligands, self-supporting stimuli- responsive gels are formed. Using microrheology, we characterized the kinetics of gelation as a function of concentration and stoichiometry of the components. We also show that such gels are effective in encapsulating and releasing small molecules. Moreover, TPR gels alone are fully compatible with cell growth, whereas gels loaded with an anticancer compound release the compound, resulting in cell death. Thus, we have demonstrated that this new class of tunable biomaterials is ripe for further development as tissue engineering and drug delivery platform. # 2012 Wiley Periodicals, Inc. Biopolymers 97: 508–517, 2012. Keywords: protein design; repeat proteins; smart gels; modular; release V V C 2012 Wiley Periodicals, Inc. 508 Biopolymers Volume 97 / Number 7