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

Abstract

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 (13)C and (15)N, and establishes the scalar connectivity pathway between nuclei through the large (1)J(NH), (1)J(CH)(, 1)J(CC), and (1)J(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 (3)J(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.

Figure 1

Dominique Marion (left) and Lewis Kay, carrying eight 5-Mb disks containing the time domain data of a single 3D ¹⁵N NOESY-HMQC experiment from the Nicolet 500 MHz spectrometer to a SUN work station, for processing with their in-house software.

Figure 2

Hardware interfaced to Dennis Torchia's Bruker AM500 console for generating a computer-controlled third and fourth channel, needed for execution of the early generation of triple resonance NMR experiments.