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A Transient and Low-Populated Protein-Folding Intermediate at Atomic Resolution

Transient Protein Conformations Transient conformations are important to protein function; however, detecting and characterizing these states is technically challenging. Korzhnev et al. (p. 1312; see the Perspective by Al-Hashimi) combined recently developed methods to determine the three-dimensional atomic-resolution structure of a transient intermediate of a four-helix bundle protein domain. The intermediate formed rapidly but, owing to structural peculiarities, slowly rearranged into its native state. The methods can be applied not only to folding intermediates but also to excited states important for protein function. Nuclear magnetic resonance and computational methods are combined to determine the structure of “invisible” excited protein states. Proteins can sample conformational states that are critical for function but are seldom detected directly because of their low occupancies and short lifetimes. In this work, we used chemical shifts and bond-vector orientation constraints obtained from nuclear magnetic resonance relaxation dispersion spectroscopy, in concert with a chemical shift–based method for structure elucidation, to determine an atomic-resolution structure of an “invisible” folding intermediate of a small protein module: the FF domain. The structure reveals non-native elements preventing formation of the native conformation in the carboxyl-terminal part of the protein. This is consistent with the kinetics of folding in which a well-structured intermediate forms rapidly and then rearranges slowly to the native state. The approach introduces a general strategy for structure determination of low-populated and transiently formed protein states.