Triple resonance three-dimensional protein NMR: Before it became a black box

Three-dimensional triple resonance experiments have become an integral part of virtually every solution NMR study of proteins. The approach relies on uniform isotopic enrichment of proteins with ¹³C and ¹⁵N, and establishes the scalar connectivity pathway between nuclei through the large ¹J _NH, ¹J _CH^{, 1}J _CC, and ¹J _CN couplings. The magnetization transfer process takes place through multiple, efficient one-bond magnetization transfer steps, rather than a single step through the smaller and variable ³J _HH couplings. The relatively large size and good uniformity of the one-bond couplings allowed the design of efficient magnetization transfer schemes that are effectively uniform across a given protein, nearly independent of conformation. Although conceptually straightforward, practical implementation of three-dimensional triple resonance experiments on proteins originally posed serious challenges. This account provides a personal perspective on some of the historical background to this work, the problems encountered as well as their solutions, and their evolution into today's standard arsenal of experiments.