NMR methods for structural biology: improved experiments and workflows

The advent of ultra-high-field NMR instrumentation and high-performance probe technology brings both unprecedented opportunities and new experimental challenges. Improving sensitivity, resolution, and acquisition time remains crucial for extending the applicability of NMR spectroscopy to complex biological systems, including intrinsically disordered proteins or regions (IDPs/IDRs), and large macromolecular assemblies. This thesis presents methodological strategies to enhance spectral quality across multiple fronts. The optimization of 13C-detected experiments at 1.2 GHz is first explored, demonstrating how IDPs/IDRs benefit from improved resolution and sensitivity, and how complementary CON variants (HN BEST CON, Hα flip CON, and standard CON) provide detailed insight into large unstructured proteins. Multiple-receiver (MR) acquisition schemes are introduced to exploit recovery delays productively, enabling simultaneous datasets acquisition and reducing experimental time without compromising data quality. Building on these principles, multiple acquisition strategies are applied to challenging systems. In particular, orthogonal isotopic labelling combined with MR acquisition enables the characterization of the trimeric MAX/Myc-BRCA1 complex. This strategy is also exploited to probe mobile and charged side chains. Several methodological approaches, including direct 13C detection, double-quantum coherence transfer, scalar-coupling-based cross polarization, and selective excitation, are integrated into combined experiments. These workflows provide complementary coverage of acidic and basic residues and are applied to map side-chain perturbations in the interaction between the SARS-CoV-2 nucleocapsid N-terminal domain and heparin. Another aspect addressed in this work is the optimization of radiofrequency pulse through Optimal Control (OC) theory. Applications range from 13C broadband decoupling in 15N-detected experiments to optimized two- and three-dimensional experiments. Finally, the potential of 15N-detected TROSY experiments is demonstrated. By integrating selective decoupling of aliphatic protons with BEST-type longitudinal relaxation enhancement, a BEST N-TROSY scheme is established, offering superior resolution without loss of sensitivity under near-physiological conditions.