Domain editor

The A39G FF domain folds to a volcano-shaped free-energy surface via separate pathways


Protein molecules interconvert between several states, and this exchange process plays an important role in function and dysfunction. Here, we use powerful NMR experiments that probe sparsely populated, transiently formed conformational states that exchange with a highly populated, stable folded conformer to elucidate the folding mechanism of a small protein domain. In particular, by analyzing the exchange profiles in detail, it becomes possible to obtain structural data for States which are approximately 10 times less populated and live less long than before. Our results establish that the small FF domain folds via two partially structured intermediates and involves a pair of discrete pathways in which the unfolded state becomes increasingly structured along a volcano-like free-energy surface.


Conformational dynamics play a critical role in protein folding, misfolding, function, dysfunction, and aggregation. While detecting and studying the different conformational states populated by protein molecules on their free energy surfaces (FES) remains a challenge, NMR spectroscopy has become an invaluable experimental tool to explore the FES of a protein, because conformational dynamics can be probed at atomic resolution. over a wide range of time scales. Here, we use chemical exchange saturation transfer (CEST) to detect “invisible” minor states on the energy landscape of the A39G mutant FF domain that exhibited “two-state” folding kinetics in traditional experiments. Although the CEST was mainly limited to process studies with rates between ∼5 and 300 s−1 involving sparse states with populations as low as ∼1%, we show that the line widening that is often associated with minor state dips in CEST profiles can be exploited to inform about additional conformers, with lifespans an order of magnitude shorter and populations close to 10-fold smaller than what is typically characterized. Our analysis of CEST profiles that exploits linewidths of minor states of the 71-residue A39G FF domain establishes a folding mechanism that can be described in terms of a four-state exchange process between interconverted states spanning two orders in magnitude from ∼100 to ∼15,000 μs. A similar folding pattern is also established for the wild-type domain. The study shows that the folding of this small domain passes through a pair of sparse and partially structured intermediates via two discrete pathways on a volcano-like FES.


    • Accepted October 1, 2021.
  • Author contributions: VPT, YT, DD, LEK and PV conducted research; Data analyzed by VPT, YT, DD, LEK and PV; and LEK and PV wrote the article.

  • The authors declare no competing interests.

  • This article is a direct PNAS submission. HJD is a guest editor invited by the Editorial Board.

  • This article contains additional information online at