Intact Transition Epitope Mapping-Force Interferences by Variable Extensions (ITEM-FIVE).

Abstract

Investigations on binding strength differences of non-covalent protein complex components were performed by mass spectrometry. T4 fibritin foldon (T4Ff) is a well-studied miniprotein, which together with its biotinylated version served as model system to represent a compactly folded protein to which an Intrinsically Disordered Region (IDR) was attached. The apparent enthalpies of the gas phase dissociation reactions of the homo-trimeric foldon F-F-F and of the homo-trimeric triply biotinylated foldon bF-bF-bF have been determined to be rather similar (3.32 kJ/mol and 3.85 kJ/mol) but quite distinct from those of the singly and doubly biotinylated hetero-trimers F-F-bF and F-bF-bF (1.86 kJ/mol and 1.08 kJ/mol). Molecular dynamics simulations suggest that the ground states of the (biotinylated) T4Ff trimers are highly symmetric and well comparable to each other, indicating that the energy levels of all four (biotinylated) T4Ff trimer ground states are nearly indistinguishable. The experimentally determined differences and/or similarities in enthalpies of the complex dissociation reactions are explained by entropic spring effects, which are noticeable in the T4Ff hetero-trimers but not in the T4Ff homo-trimers. A lowering of the transition state energy levels of the T4Ff hetero-trimers seems likely because the biotin moieties, mimicking intrinsically disordered regions (IDRs), induced asymmetries in the transition states of the biotinylated T4Ff hetero-trimers. This transition state energy level lowering effect is absent in the T4Ff homo-trimer, as well as in the triply biotinylated T4Ff homo-trimer. In the latter, the IDR-associated entropic spring effects on complex stability cancel each other out. ITEM-FIVE enabled semi-quantitative determination of energy differences of complex dissociation reactions, whose differences were modulated by IDRs attached to compactly folded proteins.