Characterizing conformational ensembles of multi-domain proteins using anisotropic paramagnetic NMR restraints

It has been over two decades since paramagnetic NMR started to form part of the essential techniques for structural analysis of proteins under physiological conditions. Paramagnetic NMR has significantly expanded our understanding of the inherent flexibility of proteins, in particular, those that are formed by combinations of two or more domains. Here, we present a brief overview of techniques to characterize conformational ensembles of such multi-domain proteins using paramagnetic NMR restraints produced through anisotropic metals, with a focus on the basics of anisotropic paramagnetic effects, the general procedures of conformational ensemble reconstruction, and some representative reweighting approaches.