Abstract
Structural dynamics of fatty acid binding proteins (FABPs), which accommodate poorly soluble ligands in the internalized binding cavities, are intimately related to their function. Recently, local unfolding of the α-helical cap in a variant of human intestinal FABP (IFABP) has been shown to correlate with the kinetics of ligand association, shedding light on the nature of the critical conformational reorganization. Yet, the physical origin and mechanism of the functionally relevant transient unfolding remain elusive. Here, we investigate the intrinsic structural instability of the second helix (αII) of IFABP in comparison with other segments of the protein using hydrogen-exchange NMR spectroscopy, microsecond molecular dynamics simulations, and enhanced sampling techniques. Although tertiary interactions positively contribute to the stability of helices in IFABP, the intrinsic unfolding tendency of αII is encoded in its primary sequence and can be described by the Lifson-Roig theory in the absence of tertiary interactions. The unfolding pathway of αII in intact proteins involves an on-pathway intermediate state that is characterized with the fraying of the last helical turn, captured by independent enhanced sampling methods. The simulations in this work, combined with hydrogen-exchange NMR data, provide new, to our knowledge, atomistic insights into the functional local unfolding of FABPs.