Using CEST NMR to discover previously unobserved states on the free energy surface of proteins: Application to the L99A cavity mutant of T4 lysozyme

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments establish that at room temperature, the L99A cavity mutant of T4 lysozyme (L99A T4L) interconverts between two compact folded conformations on the millisecond timescale. These include the native state in which the sidechain of Phe114 is exposed to solvent (E state) and a near-native minor state in which the aromatic moiety of Phe114 is buried in the core of the protein (B state, ∼2%). Molecular dynamics simulations that have captured the E to B interconversion in L99A T4L and related mutants suggest that these proteins adopt compact folded conformations other than E and B, yet extensive CPMG studies have not detected such states. In an effort to detect these more elusive conformers experimentally, we have recorded Chemical Exchange Saturation Transfer (CEST) experiments, as the widths of minor state dips in the resulting CEST profiles are sensitive to additional, even more sparse, conformers. Analysis of amide ¹⁵N and ¹³CH₃ CEST profiles recorded on L99A T4L show that in addition to states E and B, a rare state (I) populated to ∼0.2% (11.5 °C) exchanges rapidly with state B. CEST-based urea m-values establish that all three states are compact, with interconversion between them proceeding via compact transition states. This study highlights the utility of CEST to characterize the free energy surface of a protein by detecting states with a wide range of lifetimes (100 ms to 100 μs) in ways that are not possible using other relaxation-based NMR techniques.