Backbone dynamics and structural characterization of the partially folded A state of ubiquitin by H-1, C-13, and N-15 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 C-13,N-15-labeled ubiquitin. Complete backbone (CO)-C-13,C-13(alpha), N-15, and H-1(N) assignment was achieved. (CO)-C-13 and C-13(alpha) chemical shifts and H-1-H-1 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 N-15 relaxation data, indicating the presence of three loosely coupled secondary structural segments with enhanced internal mobility as compared to the native state.