NMR studies of protein dynamics and structure

................................................................................................... 9 Introduction............................................................................................. 10 Aim of this thesis........................................................................................................ 10 Adenylate kinase........................................................................................................ 11 Peroxiredoxin Q......................................................................................................... 12 The ribosomal protein S16......................................................................................... 14 Protein stability........................................................................................ 15 Protein folding and the two-state model................................................................... 15 Protein denaturation.................................................................................................. 16 The unfolded state...................................................................................................... 17 Enzyme dynamics....................................................................................................... 18 Biophysical techniques............................................................................. 22 CD spectroscopy......................................................................................................... 22 Fluorescence spectroscopy......................................................................................... 23 Stopped-flow spectroscopy........................................................................................ 24 NMR theory............................................................................................... 26 The NMR signal.......................................................................................................... 26 The rotating frame..................................................................................................... 28 The chemical shift...................................................................................................... 28 Chemical shift anisotropy.......................................................................................... 29 J couplings.................................................................................................................. 29 Dipolar couplings....................................................................................................... 30 Residual dipolar couplings......................................................................................... 30 Relaxation................................................................................................................... 31 T1 relaxation............................................................................................................... 32 T2 relaxation............................................................................................................... 32 CPMG relaxation dispersion...................................................................................... 33 Hydrogen-deuterium exchange................................................................................. 33 The NOESY experiment............................................................................................. 35 The NMR timescale.................................................................................................... 36 Assignment procedures.............................................................................................. 37