Functional Assembly of Protein Fragments Induced by Spatial Confinement

Natural proteins are often confined within their local microenvironments, such as three-dimensional confinement in organelles or two-dimensional confinement in lipid rafts on cytoplasmic membrane. Spatial confinement restricts proteins' entropic freedom, forces their lateral interaction, and induces new properties that the same proteins lack at the soluble state. So far, the phenomenon of environment-induced protein functional alteration still lacks a full illustration. We demonstrate here that engineered protein fragments, although being non-functional in solution, can be re-assembled within the nanometer space to give the full activity of the whole protein. Specific interaction between hexahistidine-tag (His-tag) and NiO surface immobilizes protein fragments on NiO nanoparticles to form a self-assembled protein "corona" on the particles inside the nanopores of mesoporous silica. Site-specific assembly forces a shoulder-by-shoulder orientation and promotes fragment-fragment interaction; this interaction together with spatial confinement of the mesopores results in functional re-assembly of the protein half fragments. To our surprise, a single half fragment of luciferase (non-catalytic in solution) exhibited luciferase activity when immobilized on NiO in the mesopores, in the absence of the complimentary half. This shows for the first time that spatial confinement can induce the folding of a half fragment, reconstitute the enzyme active site, and re-gain the catalytic capability of the whole protein. Our work thereby highlights the under-documented notion that aside from the chemical composition such as primary sequence, physical environment of a protein also determines its function.