Enhancement of Protein Stability by Quenching Millisecond Conformational Dynamics

Abstract

Protein folding may involve folding intermediates. Ubiquitin (Ub) is a 76-residue small protein essential in post-translational modification and cell signaling. Ub is also a model system of protein folding. Previous studies have indicated the involvement of a folding intermediate, as Ub C-terminal residues, including strand {beta}5, only dock correctly at a later stage. The natively folded Ub undergoes conformational dynamics over a vast range of timescales. At the millisecond timescale, Ub transiently digresses to a C-terminally retracted state, which is extremely rare and has only been recently identified at an elevated temperature. Herein through a conjoint use of NMR, MS, and MD simulations, we have established a link between Ub millisecond dynamics and protein stability. Among the alanine mutations that have been systematically introduced to the hydrophobic residues in {beta}5, L67A and L69A elevate the population of the retracted state and enhance conformational interconversion, which facilitates the undocking of {beta}5 and the exposure of protein hydrophobic core. Conversely, L71A and L73A mutations decrease the population of the retracted state and quench millisecond dynamics, which causes a significant enhancement of protein stability. As such, the transition state of Ub millisecond dynamics is the much sought-after folding intermediate, whereas C-terminal mutations alleviate the dependence on this intermediate and reduce the unfolding to an all-or-none process. Though having a negative impact on protein stability, Ub millisecond dynamics likely facilitate proper protein turnover and allow the fulfillment of its biological function.