Elsevier, Cell, 6(139), p. 1109-1118, 2009
DOI: 10.1016/j.cell.2009.11.022
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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.