Pinpoint-NMR: A sequence-specific labeling technique to accelerate NMR investigation of large proteins

The cell is an ensemble of dynamic molecular machines. NMR spectroscopy is the method of choice to observe complex conformational changes, transient interactions, and dynamics of proteins at atomic resolution. The introduction of methyl-specific labeling has made it possible to study protein assemblies of several hundred kDa by solution NMR. However, the high number of overlapping signals and the labor-intensive process of site specific analysis hamper NMR investigation of large hetero-oligomeric assemblies at room temperature. While segmental labeling or site-specific labeling offer opportunities to simplify 2D NMR spectra, the complexity of sample preparation or the characteristic low expression yield drastically limit its subsequent applications. Therefore, efficient, widely applicable, and straightforward labeling strategies are necessary to integrate solution NMR analysis of large proteins (such as monoclonal antibodies) into R&D pipelines for biomedical industry. I propose to harness the natural (anti)codon code for multi-site, sequence-specific isotope labeling. The resulting method would simplify the spectral congestion typical of 2D NMR spectra of large proteins, allowing targeted investigation of sites of interest (e.g. catalytic centers) and streamlining residue assignment. This method will reduce the time required for residue specific analysis of NMR data from months to days, thus expanding our ability to study large, medically-relevant complexes and address current biological challenges.