Nuclear magnetic resonance spectroscopy provides an ideal toolbox for elucidating structure, dynamics and interactions of biomacromolecules in solution, since the offered possibility to tune and adjust the experimental conditions, i.e. buffer components, salt concentration, additives, temperature and pH, yields realistic and atomic-resolution structures of biomacromolecules under physiological conditions. In order to study protein-protein or protein-ligand interactions as well as the positioning and conformation of flexible domains within proteins in solution, long-range structural restraints are urgently needed. Paramagnetic nuclear magnetic resonance spectroscopy, more specifically pseudocontact shifts and residual dipolar couplings generated by lanthanide chelating tags, can deliver such long-range restraints and thereby render amenable the structural analysis of large protein complexes as well as protein-ligand binding in solution.

Design of next-generation lanthanide chelating tags

In order to generate strongly paramagnetic lanthanide chelating tags that are sufficiently immobilized on the surface of the protein and generate thereby large paramagnetic effects, i.e. large pseudocontact shifts and residual dipolar couplings, we synthesize sterically overcrowded DOTA-derived lanthanide chelating tags with different linker systems. More specifically, the introduction of sterically demanding substituents, e.g. isopropyl groups, as well as the introduction of novel, reduction-stable linker moieties, e.g. pyridinethiazole derivatives, proved to be highly beneficial in order to generate lanthanide chelating tags with optimal properties and general applicability.

Applications of lanthanide chelating tags

Macromolecular function frequently requires that proteins change conformation into high-energy states1–4. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts5 (PCSs) with Carr–Purcell– Meiboom–Gill (CPMG) relaxation dispersion6 (PCS–CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS–CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.