Protein NMR Spectroscopy
Practical Techniques and Applications
Book
Hardback
416 pages
978-1-4051-6721-5 (ISBN)
Description
Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique in structural biology for obtaining high resolution 3-D structures of proteins, second only, and complementary to X-ray crystallography. Molecules are studied in solution, where conditions are closer to what is found in the cell. It is the primary technique used to obtain information on intrinsically disordered (unfolded) proteins, since these proteins will not crystallize easily. NMR provides information about conformational or chemical exchange, internal mobility and dynamics at timescales varying from picoseconds to seconds. Protein folding studies can be done by monitoring spectra of a protein in real time. NMR is also very efficient in mapping interactions with other molecules, e.g. protein/protein, protein/nucleic acid. The aim of this book is to provide the newcomer to NMR techniques with practical guidance on how to choose the right experiment, how to carry out the experiment, and how to analyse the resulting spectra. Those who are familiar with the chemical applications of NMR will also find it helpful in describing the special requirements of proteins.
More details
Language
English
Place of publication
Chicester
United Kingdom
Publishing group
John Wiley and Sons Ltd
Target group
Professional and scholarly
ISBN-13
978-1-4051-6721-5 (9781405167215)
Copyright in bibliographic data is held by Nielsen Book Services Limited or its licensors: all rights reserved.
Schweitzer Classification
Persons
Professor Gordon Roberts is Head of the School of Biological Sciences, University of Leicester. Dr Christina Redfield is Reader, Oxford Centre for Molecular Sciences, University of Oxford.
Content
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)