457514 pH-Sensitive Mechanical Properties of Elastin-like Polypeptides
ELPs are composed of a repeating pentapeptide amino acid sequence containing one “guest residue” position that can be an amino acid of choice. This guest residue allows design flexibility. The inclusion of ionizable amino acids in the guest residue position confers pH-dependent surface charges. pH sensitivity can be achieved across a wide pH range if ionizable amino acids with a range of pKa values are included. Additionally, pH sensitivity may be induced by including ionizable amino acids before or after the ELP sequence in a protein tag. We expect that pH sensitivity will persist in hydrogels made from ELPs and that hydrogels containing pH-dependent ionizable groups will swell and soften when those groups become charged. However, to our knowledge, there are not yet any studies of pH sensitivity in ELP-based hydrogels. Thus, in this study, we seek to tune the mechanical properties of ELP-based hydrogels by varying the charged guest residues and tags.
We have created a family of recombinant ELPs with ionizable guest residues and ionizable protein tags. The ionizable guest residues include tyrosine (pKa ~10.4) and lysine (pKa ~10.5). The protein tags include histidine (pKa ~6.0) and aspartic acid (pKa ~3.7). The pKa of an amino acid indicates the pH at which it becomes ionized; the charge of the amino acid side chain changes from positively charged to neutral (lysine and histidine) or from neutral to negatively charged (tyrosine and aspartic acid). These shifting and competing charges contribute to the overall charge behavior of the protein. We hypothesize that lysine and tyrosine will contribute to the pH sensitivity at pH values above 7 and that chemically blocking these guest residues will reduce the pH sensitivity above 7. We also hypothesize that histidine and aspartic acid will contribute to the pH sensitivity at pH values below pH 7, and designs that do not contain the protein tag with histidine and aspartic acid will have reduced pH sensitivity below pH 7.
We have found that ELP hydrogels containing both ionizable guest residues and protein tags are sensitive to pH change both above and below pH 7. The Young’s Modulus of these hydrogels decreased by 60% when submerged in a buffer at pH values below or above 7 compared to gels at pH 7. In buffers of pH 5 or below, the hydrogels increased in size, and gels at pH 3 were twice as swollen as gels at pH 7. When the lysine and tyrosine residues were selectively blocked through acetylation, these hydrogels lost their pH sensitivity and their Young’s Modulus did not change at pH values above 7. Overall, our conclusions demonstrate that including ionizable guest residues or protein tags creates pH sensitivity in ELP hydrogels across a wide pH range and that this sensitivity can be altered through chemical treatments. Because the pH sensitivities of the ELP sequence and the protein tag exhibit distinct behaviors, it may be possible to use ionizable protein tags in recombinant protein designs using sequences besides ELPs in order to include pH sensitivity in the resulting hydrogel. Thus, ELP hydrogels with ionizable groups may be useful in designing materials for pH-sensitive drug release in areas such as acidic tumors or in environmentally responsive microfluidic valves.