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, ¹³C^α, ¹⁵N, and ¹H^N assignment was achieved. ¹³CO and ¹³C^α 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 β-sheet and the central α-helix, the native secondary structural elements are largely conserved. The C-terminal half, which is in the native form rich in β-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.