Summary Phosphorylation is one of the most commonly used signaling mechanisms in biology. However, the molecular transition pathways between inactive and active states are poorly understood. Here we quantitatively characterize the free-energy landscape of activation of the signaling protein Nitrogen regulatory protein C (NtrC) by connecting functional protein dynamics of phosphorylation-dependent activation to protein folding. We show that only a rarely populated, pre-existing active conformation is capable of being phosphorylated. Atomistic details of a pathway for the complex conformational transition, inferred from molecular dynamics simulations (Lei et al., 2009) is experimentally tested here by NMR dynamics experiments. We found that the loss of native stabilizing contacts during activation is compensated by non-native transient atomic interactions during the transition. The results demonstrate the power of combining computation with experimental corroboration to unravel atomistic details of native-state protein energy landscapes by expanding the energy landscape from the ground states to transition landscapes.

The interconversion between inactive and active protein states, traditionally described by two static structures, is at the heart of signaling. However, how folded states interconvert is largely unknown due to the inability to experimentally observe transition pathways. Here we explore the free energy landscape of the bacterial response regulator NtrC by combining computation and NMR, and discover unexpected features underlying efficient signaling. We find that functional states are defined purely in kinetic and not structural terms. The need of a well-defined conformer, crucial to the active state, is absent in the inactive state, which comprises a heterogeneous collection of conformers. The transition between active and inactive states occurs through multiple pathways, facilitated by a number of nonnative transient hydrogen bonds, thus lowering the transition barrier through both entropic and enthalpic contributions. These findings may represent general features for functional conformational transitions within the folded state.

While ground state structures combined with chemical tools and enzyme kinetics deliver useful information on possible chemical mechanisms of enzyme catalysis, they do not unravel the finely balanced energy inventory to explain the impressive rate enhancement of enzymes. For this goal, a complete description of enzyme catalysis in the form of an energy landscape is needed. Since the rate of catalysis is determined by the climb over a sequence of energy barriers, we focus here on the critical question of transition pathways. A combination of time-resolved NMR and simulation deliver a glimpse into how proteins can so efficiently move within the ensemble of the native conformations while avoiding unfolding during that journey. The loss of energy due to breakage of native contacts is compensated by non-native transient hydrogen bonds during the transition thereby “holding on” to the energy until the new native contacts form that define the alternate functional state. The use of kinetic isotope effects (KIE) to study the chemical step show that coordinated atomic fluctuations of the protein component dictate the probability of “correct” distance and orientation, due to its extreme sensitivity to distance. The examples here stress the point that highly choreographed conformational sampling together with chemical integrity is a prerequisite for efficient enzyme catalysis.