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) c hemical shifts and H-1-H-1 nuclear Overhauser enhancement (NOE) connec tivities indicate different behavior for the N-terminal and the C-term inal halves of the protein. In the N-terminal half of the A state, com prising the antiparallel beta-sheet and the central alpha-helix, the n ative secondary structural elements are largely conserved. The C-termi nal half, which is in the native form rich in beta-strand character, u ndergoes a methanol-induced transition to a dynamic state with a unifo rmly high propensity for helical structure. This behavior is also refl ected in backbone N-15 relaxation data, indicating the presence of thr ee loosely coupled secondary structural segments with enhanced interna l mobility as compared to the native state.