Discovering Conformational Sub-States Relevant to Protein Function

Abstract

Native protein dynamics are governed by a hierarchical energy landscape, a multi-level configurational space whose hills and valleys correspond respectively to transition states and stable conformational sub-states. Internal motions enable proteins to explore this rugged landscape; increasingly, proteins are conceptualized as richly diverse ensembles rather than static structures. But the role of conformational fluctuations (or multiple conformations) in designated function of proteins is widely debated. Recent evidence indicates that sub-states of protein conformations exist containing both structural and dynamical features important for function. The low populations in these sub-states and the transient nature of conformational transitions have presented significant challenges for their identification and characterization. To overcome this challenge we have developed quasi-anharmonic analysis (QAA). QAA utilizes higher-order statistics of protein motions allowing identification of various states in the conformational hierarchy; further, the focus on anharmonicity allows the identification of conformational transitions between sub-states. QAA of equilibrium simulations of human ubiquitin and T4 lysozyme, elucidates a hierarchy of functionally relevant sub-states and protein motions involved in molecular recognition. In combination with a reaction pathway sampling method, QAA allows characterization of conformational sub-states associated with an enzyme reaction such as the cis/trans isomerization of peptidyl-prolyl amide bonds catalyzed by the enzyme cyclophilin A. In all three cases QAA reveals presence of a number of conformational sub-states at different levels in the hierarchy, with specific sub-states containing crucial structural and dynamical elements relevant for identification of binding other proteins (ubiquitin), binding substrate (lysozyme) and enzyme-substrate interactions in the active-site for the transition state formation and reaction mechanism (cyclophilin A). Overall, QAA provides a novel framework to intuitively understand biophysical basis of conformational diversity and its relevance to protein function.