This article presents the de novo design of pH-responsive self-assembling helical protein filaments. The researchers designed protein subunits containing six or nine buried histidine residues that assemble into micrometre-scale, well-ordered fibres at neutral pH. The cryogenic electron microscopy structure of an optimized design closely matches the computational design model. The filaments undergo a sharp and reversible transition from assembled to disassembled states over a 0.3 pH unit range, with rapid disassembly occurring within less than 1 second upon a drop in pH. The transition midpoint can be tuned by modulating buried histidine-containing hydrogen bond networks. The study demonstrates that computational protein design can create unbound nanomaterials that rapidly respond to small pH changes. The researchers designed pH-responsive filaments by connecting subunits with short loops to break internal symmetry and allow asymmetric interfaces to drive fibre assembly. They generated 45,000 helical filament backbones and selected 18 designs for experimental testing. These designs were expressed in E. coli and purified. Two designs formed filaments, as seen in negative stain electron microscopy images. Structural characterization using cryogenic electron microscopy revealed that the filaments have a close match between the design model and the cryo-EM structure. The pH transition midpoint can be modulated by the number of buried histidines. The researchers also characterized the pH response of the filaments using negative stain EM and fluorescence microscopy. The filaments showed a sharp pH transition over 0.3 pH units, with rapid disassembly upon pH drop. The pH response was further studied using liquid-phase atomic force microscopy, revealing a sharp transition over 0.1 pH units and a steep increase in disassembly kinetics over 0.3 pH units. The study also explored the use of photoacid to induce pH changes and disassemble the filaments. The results show that the pH response mechanism can be harnessed for external stimulus-driven response. The researchers concluded that the ability to generate micrometre-scale pH-responsive filaments is an advance in computational design of environmentally responsive protein nanomaterials. The two fibre systems described exhibit remarkable and tunable pH dependence of disassembly. The study highlights the potential of these filaments for applications in drug delivery and other fields. The researchers also discussed the limitations of their approach and the potential for future improvements using deep learning protein design methods.This article presents the de novo design of pH-responsive self-assembling helical protein filaments. The researchers designed protein subunits containing six or nine buried histidine residues that assemble into micrometre-scale, well-ordered fibres at neutral pH. The cryogenic electron microscopy structure of an optimized design closely matches the computational design model. The filaments undergo a sharp and reversible transition from assembled to disassembled states over a 0.3 pH unit range, with rapid disassembly occurring within less than 1 second upon a drop in pH. The transition midpoint can be tuned by modulating buried histidine-containing hydrogen bond networks. The study demonstrates that computational protein design can create unbound nanomaterials that rapidly respond to small pH changes. The researchers designed pH-responsive filaments by connecting subunits with short loops to break internal symmetry and allow asymmetric interfaces to drive fibre assembly. They generated 45,000 helical filament backbones and selected 18 designs for experimental testing. These designs were expressed in E. coli and purified. Two designs formed filaments, as seen in negative stain electron microscopy images. Structural characterization using cryogenic electron microscopy revealed that the filaments have a close match between the design model and the cryo-EM structure. The pH transition midpoint can be modulated by the number of buried histidines. The researchers also characterized the pH response of the filaments using negative stain EM and fluorescence microscopy. The filaments showed a sharp pH transition over 0.3 pH units, with rapid disassembly upon pH drop. The pH response was further studied using liquid-phase atomic force microscopy, revealing a sharp transition over 0.1 pH units and a steep increase in disassembly kinetics over 0.3 pH units. The study also explored the use of photoacid to induce pH changes and disassemble the filaments. The results show that the pH response mechanism can be harnessed for external stimulus-driven response. The researchers concluded that the ability to generate micrometre-scale pH-responsive filaments is an advance in computational design of environmentally responsive protein nanomaterials. The two fibre systems described exhibit remarkable and tunable pH dependence of disassembly. The study highlights the potential of these filaments for applications in drug delivery and other fields. The researchers also discussed the limitations of their approach and the potential for future improvements using deep learning protein design methods.