Nuclear magnetic resonance (NMR) spectroscopy analyses molecular structures through the way nuclear spins respond to magnetic fields and radio waves. Wüthrich established the “sequential assignment” strategy that matches each spectral peak to a specific hydrogen atom in the protein chain, enabling complete three-dimensional structures in solution. His approach feeds distance restraints derived from multidimensional spectra into distance-geometry calculations to rebuild the molecule’s shape. Because crystallisation is unnecessary, proteins can be examined under near-physiological conditions, revealing both structure and motion. The technique is now widely used for validating drug targets and studying soluble misfolded proteins such as those involved in prion diseases.

In multidimensional NMR, correlation experiments such as COSY and NOESY visualise scalar couplings and spatial proximities, providing conformational restraints. Wüthrich’s sequential assignment protocol tracks H-N, Cα and Cβ chemical shifts along the sequence while cross-checking NOE patterns, ultimately identifying every residue. NOE distance restraints, dihedral angles and relaxation data are then fed into simulated annealing to generate structure ensembles whose convergence is assessed by RMSD. Solution NMR captures side-chain fluctuations and folding intermediates, complementing the static snapshots of X-ray crystallography. Recent advances such as TROSY and tailored isotope labelling extend the method to complexes exceeding 50 kDa.

Wüthrich systematised cross-peak analysis of 2D/3D NOESY spectra and spearheaded the development of NOE-driven distance-geometry algorithms such as DIANA/CYANA. Sequential assignment employs HNCO→HNCACB→CBCA(CO)NH 3-D experiments to enable backbone walks. Distance and angle restraints are optimised through molecular dynamics against a target function, and synthetic RDC inclusion permits orientation-matrix evaluation. His framework has been applied to allosteric network mapping in solution, low-affinity ligand screening and micelle-embedded membrane-protein structure determination, underpinning NMR-based structural genomics.