Physics

Kinetics

Crystallography

Biology

Biophysics

Energy landscape

Chemistry

Quantum mechanics

Biochemistry

Nature

Macromolecular function frequently requires that&nbsp;proteins change&nbsp;conformation into high-energy states1–4. However, methods for solving the structures of these functionally essential, lowly populated states&nbsp;are lacking. Here we develop a method for high-resolution structure determination of minorly&nbsp;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&nbsp;(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. Combining&nbsp;NMR spectroscopy-derived pseudocontact shifts (PCSs)&nbsp;with Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion enables protein structure determination&nbsp;of lowly populated high-energy states&nbsp;that are essential for macromolecular function.

Structure determination of high-energy states in a dynamic protein ensemble