Exploring the free energy surface of proteins using CEST NMR experiments

Protein molecules interconvert between multiple conformational states that can play important roles in their function, misfunction and folding. Often these states are invisible to traditional biophysical methods as they are sparsely populated. The Chemical Exchange Saturation Transfer (CEST) NMR experiment that was originally conceived 60 years ago [1] was only relatively recently adapted to study sparsely populated states in proteins [2]. In this talk I will present a recent development that considerably extends the ability of the CEST experiment to probe exchange between multiple states over a range of timescales and show that it now becomes possible to detect invisible states with extremely low populations (~0.1%) and a wide range of lifetimes (~10-5 to ~10-1 s) [3]. Using this strategy we discovered that the small 71 residue A39G FF domain folds from the unfolded (U) to the folded (F) state via two pathways involving two intermediates (I1 and I2) on a volcano shaped free energy surface [3]. The structure of the I1 state was determined almost a decade ago using CPMG experiments [4] and we have now used CEST experiments to determine the structure of the newly discovered I2 state that turns out to be more compact than the I1 state. Urea m-values of the different states (U, I1 and I2) and the transition states that separate them have also been determined. The structures of the two folding intermediates along with the thermodynamic parameters provide unprecedented insights into the folding mechanism of the small FF domain. I will all also present recent developments with the CEST methodology that makes it ‘straight-forward’ to study three-state exchange on occurring on the millisecond time-scale thus detecting states missed by CPMG [5]. An extension of CEST to study exchange occurring at ~104 s-1 will be also be discussed [6].
[1]J Chem Phys, 1963. 39(11): p. 2892-2901.
[2]J Am Chem Soc, 2012. 134(19): p. 8148-61.
[3]Proc Natl Acad Sci U S A, 2021. 118(46): e2115113118 [4]Science, 2010. 329(5997): p. 1312-1316
[5]J Biomol NMR (in press)
[6]J Biomol NMR, 2023. 77: p. 165–181