Extending the size limit of protein nuclear magnetic resonance

In the past 15 years, nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology (1, 2). It not only is capable of solving protein structures to atomic resolution but also has the unique ability to accurately measure the dynamic properties of proteins and to probe the process of protein folding (3, 4). However, a major drawback of macromolecular NMR is its size limitation caused by two technical barriers. First, larger molecules have slower tumbling rates and shorter NMR signal relaxation times. This reduces the sensitivity of the complicated pulse sequences that often use long delays for the necessary coherence transfer steps. The increased molecular weight also introduces more complexity to a given spectrum, simply because there are more NMR-active nuclei and, therefore, more interactions among them. The current size limit of protein NMR is ≈35 kDa, but recent advances in both hardware and experimental design promise to allow the study of much larger proteins (2). The future is even brighter with the development of novel strategies for isotopic labeling of proteins that are synergistic with the new NMR techniques. One such strategy is the segmental protein isotopic labeling scheme described by Xu et al. in this issue of the Proceedings (5). Xu et al. have successfully ligated together two independently expressed, folded protein domains under mild conditions in vitro, making it possible to selectively label a given fragment or domain of modular proteins (5). They have done so by taking advantage of protein splicing (6, 7). A small number of proteins are made as precursors that contain motifs called inteins. During the maturation process, inteins are excised from these precursors, and the two resulting fragments or exteins then rejoin. This process has been called protein splicing presumably because …