Segmental and site-specific isotope labelling strategies for structural analysis of posttranslationally modified proteins

Posttranslational modifications can alter protein structures, functions and locations, and are important cellular regulatory and signalling mechanisms. Spectroscopic techniques such as nuclear magnetic resonance, infrared and Raman spectroscopy, as well as small-angle scattering, can provide insights into the structural and dynamic effects of protein posttranslational modifications and their impact on interactions with binding partners. However, heterogeneity of modified proteins from natural sources and spectral complexity often hinder analyses, especially for large proteins and macromolecular assemblies. Selective labelling of proteins with stable isotopes can greatly simplify spectra, as one can focus on labelled residues or segments of interest. Employing chemical biology tools for modifying and isotopically labelling proteins with atomic precision provides access to unique protein samples for structural biology and spectroscopy. Here, we review site-specific and segmental isotope labelling methods that are employed in combination with chemical and enzymatic tools to access posttranslationally modified proteins. We discuss illustrative examples in which these methods have been used to facilitate spectroscopic studies of posttranslationally modified proteins, providing new insights into biology.