Assembly of an artificial protein hydrogel through leucine zipper aggregation and bisulfide bond formation

We present a strategy to stabilize artificial protein hydrogels through covalent bond formation following physical association of terminal leucine zipper domains. Artificial proteins consisting of two terminal leucine zipper domains and a random coil central domain form transient networks above a certain concentration, but the networks dissolve when placed in excess buffer. Engineering of a cysteine residue into each leucine zipper domain allows formation of disulfide bonds templated by leucine zipper aggregation. Circular dichroism spectra show that the zipper domains remain helical after cysteine residues and disulfide bonds are introduced. Asymmetric placement of the cysteine residues in the leucine zipper domains suppresses intramolecular disulfide bonds and creates linked "multichains" composed of ca. 9 protein chains on average, as determined by multiangle light scattering measurements. These "multichains" act as the building units of the physical network formed by leucine zipper aggregation. The increased valency of the building units stabilizes the hydrogels in open solutions, while the physical nature of their association allows the reversibility of gelation to be retained. The gel networks dissolve at pH 12.2, where the helicity of the leucine zipper domains is reduced by ca. 90%, and re-form upon acidification. The hydrogels show anisotropic swelling when anchored on aminated surfaces and may find applications in tissue engineering, controlled release, and microarray technologies on the basis of their stability, reversibility, and swelling behavior.