Proteins and nucleic acids are not static molecules, but dynamically interconverting between multiple conformations over a range of timescales. It has become increasingly apparent that sparsely populated or transiently formed states can regulate many biological processes, including molecular recognition, enzyme catalysis, and (mis)folding. The low populations and fleeting lifetimes of these states, however, render them ‘invisible’ to standard biophysical approaches where structural characterization remains exceedingly difficult. By contrast, NMR spectroscopy is exquisitely sensitive to lowly populated conformations and can characterize their structures and dynamics at the atomic level.

  For conformations that interconvert with the ground state, advanced NMR methodology enables the detection and characterization of states with fractional populations as low as 0.5% and lifetimes ranging from microseconds to milliseconds. Powerful NMR experiments that provide atomic-level insight into lowly populated states have been designed to exploit chemical exchange, such as Carr-Purcell-Meiboom-Gill (CPMG) and rotating-frame (R_1ρ) relaxation dispersion, chemical exchange saturation transfer (CEST), and paramagnetic relaxation enhancements (PREs). Key to the success of these experiments has been the synergistic development of novel NMR pulse sequences, suitable isotope labeling schemes, and innovative computational analyses. This collection aims to disseminate methods in solution- and solid-state NMR that have enabled the detection and characterization of lowly populated states, including protocols related to sample preparation, isotope labeling schemes, pulse sequences, and data analysis. The methods collection will be a continually growing resource for researchers interested in biomolecular NMR and structural biology.