NMR Spectroscopy of Large Proteins

Abstract

Traditionally, the major obstacle to using NMR spectroscopy to gain meaningful structural information about proteins of mass greater than around 25kDa has been the poor quality of spectra. In this chapter, we discuss inherent ways and recent advances in NMR methodology that can be used to circumvent or overcome the problem of poorly resolved spectra. For example, some large proteins have functionally significant regions of inherent flexibility that are observable by NMR spectroscopy. In the case of large proteins with intrinsic paramagnetic centres, e.g. haem proteins, significant structural information can be gleaned from the hyperfine shifts of resonances out of the diamagnetic envelope. Examples from both of these situations are presented. Advances in isotopic labelling are described which have increased the upper size limit of proteins to be studied by NMR. Transverse relaxation-optimized spectroscopy (TROSY) is a method that enables the acquisition of quality spectra for large proteins and therefore has the potential to revolutionize the study of these systems. Accordingly, the latest developments in the use of TROSY methods are reviewed. Recent advances are described in the use of partial alignment of proteins to determine residual dipolar couplings as additional input restraints for the structure determination of large proteins. Finally, the use of the latest software packages to assign NMR spectra and calculate and refine protein structures is discussed.