Backbone dynamics and structural characterization of the partially folded A state of ubiquitin by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy.

Structure and dynamics of the partially folded A state of ubiquitin in a 60%/40% methanol/water mixture at pH 2 was studied by two- and three-dimensional nuclear magnetic resonance spectroscopy (NMR) using fully 13C,15N-labeled ubiquitin. Complete backbone 13CO, 13Calpha, 15N, and 1HN assignment was achieved. 13CO and 13Calpha chemical shifts and 1H-1H nuclear Overhauser enhancement (NOE) connectivities indicate different behavior for the N-terminal and the C-terminal halves of the protein. In the N-terminal half of the A state, comprising the antiparallel beta-sheet and the central alpha-helix, the native secondary structural elements are largely conserved. The C-terminal half, which is in the native form rich in beta-strand character, undergoes a methanol-induced transition to a dynamic state with a uniformly high propensity for helical structure. This behavior is also reflected in backbone 15N relaxation data, indicating the presence of three loosely coupled secondary structural segments with enhanced internal mobility as compared to the native state.