Single-Residue Mutation Switch Reconfigures the Hierarchical Structure and Assembly of Amphiphilic Protein Block Copolymers for Hydration Layer-Dominated Water-Responsive Actuation.

Protein folding dynamics emerge from subtle changes in intramolecular interactions, where hydration shapes free energy landscapes and influences conformational ensembles and supramolecular assembly. While these shape-shifting principles have been primarily explored in well-behaved systems with sharp transitions, their applicability to multidomain protein assemblies, particularly those containing partially folded or disordered regions, remains unclear. Here, we investigate how a single leucine-to-alanine mutation modulates the hierarchical interplay between folding, micelle formation, and macroscopic biomaterial behavior using two diblock protein block copolymers (BCPs): CE and CL44AE. In solution, both BCPs exhibit spectral features consistent with their C/CL44A monoblocks, with CE showing α-helical features and CL44AE more disordered ones. Upon dehydration, however, both systems display increased structural order, with CE favoring β-sheets and CL44AE adopting α-helical content. At the mesoscale, both BCPs assemble into amphiphilic micelles, but CE forms smaller, densely packed micelles, whereas CL44AE generates larger micelles that template film networks of varying pore size depending on their packing density. These differences lead to distinct solvation behavior, with CL44AE exhibiting greater uptake of predominantly mobile water, approximately twice that of CE, culminating in an actuation energy density of 2,043 kJ/m3, the highest reported for protein BCPs to date. Despite this increased uptake, CL44AE shows reduced mechanical relaxation and a rougher DSC/TGA thermogram, indicative of a broader conformational ensemble compared to CE. Comparisons to monoblocks (C, CL44A, E) and previously reported triblocks (CEC, CL44AECL44A) further highlight the importance of asymmetric diblock architectures in promoting hydration-sensitive assemblies, laying the rational design groundwork for future high-performance, water-responsive protein biomaterials.