Exploring the Effects and Interplay of Elastin-like Polypeptide (ELP) Charge and Hydrophobicity on Mcherry-ELP Fusion Protein Self-Assembly

Enzymes are attractive catalysts for a variety of applications including pharmaceutical synthesis, commodity chemical production, and biosensors due to their substrate specificity, high catalytic rates, sustainable source, and their ability to operate at mild reaction conditions. However, fabrication of enzymes into biofunctional devices requires incorporation of the protein into a material. Block copolymers are well-recognized for their ability to self-assemble into a variety of different morphologies depending on temperature and block volume fraction. It has been established that similar phase behavior is observed when polymers are conjugated to globular proteins and, importantly, that the globular proteins maintain their original function. Recent work in the Olsen lab has shown that similar self-assembly occurs in elastin-like protein (ELP)-mCherry fusion protein systems, where ELP serves as a structural coil-like block and mCherry serves as a model globular protein. These fusion protein systems are of great interest due to the advantages they offer over polymer-protein conjugates of improved ease of synthesis and purification. However, there is little understanding of how protein design can be used to control self-assembly in these fusion protein systems. Furthermore, because the entire fusion protein is technically a single poly(amino acid), understanding of how amino acid identity changes propensity for microphase separation will inform the fundamental understanding of the physics of this self-assembly process.

In this work, we systematically explored the impact of the charge and hydrophobicity of the coil-like ELP on fusion protein self-assembly. This was achieved by varying the composition of amino acids in the ELP pentapeptide repeat sequence—Xaa-Pro-Gly-Yaa-Gly (XPGYG)—in which the first position, Xaa, can be either valine (V) or isoleucine (I), with isoleucine being more hydrophobic, and the fourth position, Yaa, can be any amino acid except proline. In this work, we used a five pentapeptide repeat sequence that varied hydrophobicity by tuning the I/V content in the first position and phenylalanine (F)/V content in the fourth position. Additionally, three different charge states (neutral, negatively charged, and zwitterionic) were studied by changing some of the amino acids in the fourth position to lysine (K) and/or glutamic acid (E). Combination of all possible permutations of charge and hydrophobicity generated 9 ELP sequences that were then fused to mCherry via genetic engineering. The self-assembly of these systems was studied using small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and light scattering.