Integrating Non-NMR Distance Restraints to Augment NMR Depiction of Protein Structure and Dynamics

Nuclear magnetic resonance (NMR) structure refinement is inherently integrative. The refinement incorporates a multitude of experimental data and minimizes the difference between observed and calculated values. Here, we review how the integrative use of non-NMR measurements, in particular, distance restraints from Förster resonance energy transfer and cross-linking coupled with mass spectrometry, can augment NMR depiction of protein structure and dynamics. Refinement against both NMR and non-NMR distance restraints helps to characterize the structures of high-molecular-weight proteins and protein complexes. When a protein fluctuates among multiple conformations at millisecond or a faster timescale, NMR signals from the different conformational states may coalesce into a single set of peaks. The integration of non-NMR distance restraints facilitates the deconvolution of NMR observables to state-specific restraints. Furthermore, the integrative use of fluorescence measurements, which provides an assessment of both length scale and timescale of protein dynamics simplifies protein ensemble structure refinement otherwise with NMR restraints alone and affords a more wholesome picture of protein dynamics. Together, distance measurements are intuitive and easy to implement by using an appropriate pseudoenergy function. Future development shall involve more accurate modeling of paramagnetic and fluorescent probes, incorporation of sparse restraints from new techniques, and characterization of protein structures in a complex cellular environment.