'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