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44235-G7
Self-Assembling, Dynamic Hydrogels Cross-Linked via Complementary DNA Interactions
Summary: Natural proteins perform a variety of functions in biological systems, including actuation, catalysis, structural support, and molecular sequestering. The variety of natural protein functions suggest that they could serve as valuable and versatile building blocks for synthesis of functional materials. A function that has not been explored extensively in materials science is the ability of proteins to undergo complex conformational changes. Over 200 conformational changes are well-characterized, representing a vast unexplored databank of molecular building blocks for design of dynamic materials. To that end, our recent work supported by this PRF award involves assembly of protein-based, dynamic materials using a novel photochemical approach. This approach allows for high concentrations of protein to be functionally included into a network in response to light, and the resulting materials undergo striking volume changes upon protein-ligand binding. Photochemical assembly also enables spatial control over the location of dynamic proteins in a hydrogel network, which is likely to be important in potential materials science and engineering applications.
Approach: Strategies for direct use of biological motions to build novel materials require a mechanism to scale nanometer-scale conformational changes into macroscopic effects. To exploit molecular motions in a macroscopic material we have utilized the well-characterized conformational dynamics of the protein calmodulin (CaM). Calmodulin has two distinct conformational states. In the presence of calcium, CaM is in an extended conformation and is a dumbbell-shaped protein. Calcium-bound CaM undergoes a rapid transition from an extended dumbbell to a collapsed conformation in response to binding of ligands, which include the small molecule drug trifluoperazine (TFP). We hypothesized that appropriate incorporation of CaM into a hydrogel network would enable its motion to be translated into a spatially defined structural change in a polymer network, resulting in a protein-based, dynamic material. To assemble protein-based materials using a photochemical approach, we first generated photo-reactive protein units using an engineered protein and a Michael-type addition reaction. We synthesized and purified an engineered version of CaM that includes cysteine residues in place of tyrosine residues at the ends of the dumbbell-shaped protein (CaM (T34C, T110C)). The distance separating the two cysteine residues is approximately 50Å in the extended conformation but is reduced to approximately 15Å in the collapsed conformation. This engineered protein was reacted with a 10X molar excess of 575Da poly(ethylene glycol) diacrylate (PEGDA) chains, resulting in Michael-type addition of the sulfhydryl group on CaM's cysteines to the vinyl groups on PEGDA's acrylate termini. The resulting solution was purified, resulting in a pure PEG-CaM-PEG molecule terminated on each end with a photo-reactive group.
Results: Engineered PEG-CaM-PEG conjugates were photo-crosslinked into hydrogel networks, and over 90% of the initially included protein remained in each hydrogel during 24 hours of swelling, confirming that protein incorporation into the networks was efficient. To determine whether protein-based networks were functional, we examined their dynamic properties. Transfer of CaM-based hydrogels from an environment favoring an extended protein conformation (Ca++ buffer) to an environment favoring a collapsed conformation (Ca++ buffer with TFP ligand) resulted in a significant decrease in hydrogel volume. The amplitude of the volume decrease was dependent on the total amount of protein incorporated into the hydrogel, and hydrogels containing the largest amount of protein showed a 65% volume decrease. The hydrogels also recovered their initial volume when returned to a Ca++ buffer, indicating that changes in network properties were reversible. It is noteworthy that the dynamic range of the materials we have generated (up to 65% decrease in volume) is more than 3-fold greater than the range demonstrated in previous studies of protein-based, dynamic hydrogels (<20% change in volume).
Results also indicate that these materials can be cycled repeatedly between extended and collapsed volumes, and our photochemical approach allows for creation of heterogeneous materials, in which dynamic properties can be confined to specific locations. We used patterned photo-crosslinking to create networks containing a dynamic, CaM-containing portion and a static, CaM-free portion. When samples prepared in an environment favoring extended CaM were transferred to an environment favoring collapsed CaM, the protein-containing portion of the hydrogels displayed a significant volume change, while the protein-free PEG hydrogel did not change its volume. To test whether the dynamic property of the CaM could be utilized to apply force to its surroundings, we created another composite hydrogel, in which a CaM-based dynamic portion was surrounded by a static PEG-based portion. When the composite hydrogel was transferred to an environment containing the TFP ligand, the shrinking of the dynamic portion in the center resulted in translational motion of the static surrounding portion. Taken together, these data demonstrate that the response of protein-based dynamic hydrogels can be spatially defined and can do work, and these capabilities could be important in a variety of applications of these materials, including microfluidics, soft robotics, and drug delivery.
