Segmental Isotopic Labeling of a Central Domain in a Multidomain Protein by Protein Trans-Splicing Using Only One Robust DnaE Intein

摘要

Many proteins, including cellular signaling proteins, cell-surface receptors, and modular enzymes, are constructed from individual domains and connecting linkers. Structural analysis by NMR spectroscopy or by X-ray crystallography often focuses on self-contained domains excised from full-length proteins so as to reduce the molecular weight to a manageable size for high-quality NMR spectra or to improve crystallization by removing disordered regions. Such minimization often neglects functional aspects of a domain in an intact protein and may not represent all aspects of the structure-function relationship in the full-length context. While the structure determination of individual, isolated domains has been tremendously accelerated-in part through several structural genomics consortia-understanding of domain-domain interactions within a multidomain protein is lacking behind. NMR spectroscopy is an ideal tool for characterizing such, often transient, interactions. However, NMR spectroscopic analysis of large proteins often suffers from signal overlap. This problem becomes even more profound when proteins contain recurring modular domains and/or highly disordered regions. One solution to this overlap problem is segmental isotopic labeling that enables the exclusive incorporation of NMR active, stable isotopes into selected domains or parts of larger proteins, thereby reducing the complexity of the NMR spectra.