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

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.