1. Introduction. Aims & limitations. 2. Sample preparation, data collection & processing. Sample preparation - concentration, ionic strength, pH/pD. Basics of data collection - numbers of data points, recycle delay, pulse calibration, chemical shift referencing. Fast data collection methods (brief). Data processing - NMRPipe, apodization, linear prediction. Gordon Roberts (Leicester) + AN Other. 3. Isotope labelling. Objectives of isotope labelling - to enable heteronuclear NMR (13C and 15N) and to achieve line narrowing (2H). General labelling - 15N, 13C and 2H in E. coli, yeast and cell-free systems. Selective labelling - e.g., by amino acid type - to achieve spectral simplification. Biosynthetic selective labelling - e.g. methyl groups. SAIL labelling. Domain selective labelling (inteins). Masatasune Kainosho (Tokyo) or Kevin Gardner (Dallas). 4. Resonance assignments. NOE-based 1H assignments for small proteins and in 15N-labelled proteins (emphasis on 15N-edtied methods). Triple resonance scalar-coupling based assignment approaches. Simple TROSY-based approaches. Approaches to resonance assignment when the crystal structure is known. Automation of resonance assignment. Christina Redfield (Oxford). 5. Measurement of structural constraints. NOEs - assignments, (semi-)quantitation to give distance constraints. Chemical shifts - CSI and TALOS methods for generating f/y constraints. Spin-spin coupling constants - f/y and c1 torsion angle constraints. Residual dipolar couplings. Hydrogen bonds - H/D exchange and HNCO methods. Stefan Grzesiek (Basel), Michael Sattler (Heidelberg) or Martin Blackledge (Grenoble). 6. Calculation of structures from NMR constraints. To include a variety of approaches - e.g., XPLOR/CNS, CYANA, ARIA; Cartesian and torsion angle based systems. Dealing with ambiguous data (NOE assignments). Structural refinement. Quality checks on structures. Dealing with homodimers. Michael Nilges (Paris), Martin Blackledge (Grenoble) or possibly Alexandre Bonvin (Utrecht). 7. Relaxation and protein dynamics. Frequency/time ranges. Measurement of relaxation parameters T1, T2, NOE, T1r. Interpretation: 'model-free', spectral density mapping, etc. Relaxation dispersion (CPMG etc.). Cross correlation etc. Arthur Palmer (New York) or Jorn Werner (Southampton). 8. Structural and dynamic information on ligand binding and macromolecular complexes. Relevantexchange effects on NMR spectra. Identifying appropriate sample conditions; designing experiments for fast exchange and slow exchange. Titration experiments - Kd, exchange rates, stoichiometry, intermediates. Chemical shift mapping. Exchange-dependent experiments - STD, transferred NOE &c. Selective isotope labelling of components of complex; dealing with homo-oligomers. Isotope-edited & -filtered experiments. NMR screening experiments. Gordon Roberts (Leicester). 9. NMR of large proteins. Special isotope labelling schemes - e.g. methyl labelling (avoiding repetition of Chapter 3). Special pulse sequences - TROSY, CRINEPT, etc. Membrane proteins in micelles. Gerhard Wider (Zurich) or Lewis Kay (Toronto). 10. Paramagnetic proteins. Assignments in paramagnetic proteins - differences and difficulties from diamagnetic proteins - Distinction between regions close to and distant from the paramagnetic centre. Paramagnetic constraints - relaxation & shifts. Modifications to structure determination protocols for paramagnetic proteins. Lucia Banci / Ivano Bertini (Florence) (or possibly John Markley ( Madison)). 11. Structural information on disordered proteins. Molten globules vs. natively unfolded proteins. Special problems of disordered proteins - assignments. Interpretation of 'averaged' structural constraints. Identification & characterisation of (partially) ordered states with low population. Harald Schwalbe (Frankfurt), Flemming Poulsen (Copenhagen) or Lorna Smith (Oxford)
