Enzymatic Encoding of Topology in an Intrinsically Disordered Single‐Chain Protein

ABSTRACT Controlling the three‐dimensional topology of single‐chain nanoparticles (SCNPs) remains a central challenge in polymer and protein chemistry, particularly for intrinsically disordered systems lacking defined secondary structure. Here, we demonstrate that selective enzymatic intramolecular cross‐linking can encode topologically biased interactions in an intrinsically disordered protein (IDP), yielding compact SCNPs with reproducible cavity architecture. Using β‐casein‐rich sodium caseinate (βNaCn) as a model surrogate for bovine β‐casein (β‐Cn), microbial transglutaminase (mTGase) introduces sparse, sequence‐resolved glutamine‐lysine isopeptide bonds that drive reproducible chain collapse without inducing secondary structure. Analyses by size exclusion chromatography with quintuple detection (SEC‐D5), cross‐linking mass spectrometry (XL‐MS), molecular dynamics (MD) simulations, and SEC coupled to synchrotron small‐angle x‐ray scattering (SEC‐SAXS) converge to reveal a topology combining a stable, compact, hydrophobic core with flexible, disordered loops. These cavities are probed using Nile red (NR) fluorescence and SEC‐SAXS, which together provide topology information via guest‐induced density redistribution after NR capture. This work establishes that sparse enzymatic constraint installation, combined with residue‐resolved cross‐link mapping and orthogonal structural analysis, can encode and validate topology in a disordered single chain, thereby placing IDP‐like covalent folding in direct conceptual continuity with SCNP design.