Protein NMR Techniques

When I was asked to edit the second edition of Protein NMR Techniques, my first thought was that the time was ripe for a new edition. The past several years have seen a surge in the development of novel methods that are truly revolutionizing our ability to characterize biological macromolecules in terms of speed, accuracy, and size limitations. I was particularly excited at the prospect of making these techniques accessible to all NMR labs and for the opportunity to ask the experts to divulge their hints and tips and to write, practically, about the methods. I commissioned 19 chapters with wide scope for Protein NMR Techniques, and the volume has been organized with numerous themes in mind. Chapters 1 and 2 deal with recombinant protein expression using two organisms, E. coli and P. pastoris, that can produce high yields of isotopically labeled protein at a reasonable cost. Staying with the idea of isotopic labeling, Chapter 3 describes methods for perdeuteration and site-specific protonation and is the first of several chapters in the book that is relevant to studies of higher molecular weight systems. A different, but equally powerful, method that uses molecular biology to edit the spectrum of a large molecule using segmental labeling is presented in Chapter 4. Having successfully produced a high molecular weight target for study, the next logical step is data acquisition. Hence, the final chapter on this theme, Chapter 5, describes TROSY methods for stru- ural studies.

Includes supplementary material: sn.pub/extras

Klappentext
Carrying on in the tradition of its much praised earlier edition, A. Kristina Downing has selected new protocols describing the many recent advances in NMR methodology that are revolutionizing the field. Described in step-by-step detail by established hands-on experts, these readily reproducible techniques include methods for high-level recombinant protein expression using sophisticated isotopic labeling strategies, TROSY methods for the study of structure and dynamics, and methods for the acquisition and interpretation of residual dipolar coupling data. There are also applications of dynamics measurements on multiple timescales, new developments in NMR data analysis, structure calculation protocols, and a comprehensive chapter on solid-state methods for the study of membrane proteins. The protocols follow the successful Methods in Molecular Biology™ series format, each offering detailed laboratory instructions, an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.
State-of-the-art and highly practical, Protein NMR Techniques, 2nd Ed., makes accessible to all NMR laboratories proven techniques that are powerfully transforming the speed, accuracy, and size limit of our ability to characterize biological macromolecules.

Inhalt
Screening and Optimizing Protein Production in E. coli.- Isotopic Labeling of Recombinant Proteins from the Methylotrophic Yeast Pichia pastoris.- Perdeuteration/Site-Specific Protonation Approaches for High-Molecular-Weight Proteins.- Segmental Isotopic Labeling for Structural Biological Applications of NMR.- TROSY-Based Correlation and NOE Spectroscopy for NMR Structural Studies of Large Proteins.- Media for Studies of Partially Aligned States.- Residual Dipolar Couplings in Protein Structure Determination.- Projection Angle Restraints for Studying Structure and Dynamics of Biomolecules.- Characterizing Domain Interfaces by NMR.- Characterization of the Overall Rotational Diffusion of a Protein From 15N Relaxation Measurements and Hydrodynamic Calculations.- TROSY-Based NMR Experiments for the Study of Macromolecular Dynamics and Hydrogen Bonding.- Measurement of Intermediate Exchange Phenomena.- NMR Studies of Partially Folded Molten-Globule States.- Structure Determination of Protein Complexes by NMR.- NMR Studies of Protein-Nucleic Acid Interactions.- Using NMRView to Visualize and Analyze the NMR Spectra of Macromolecules.- Automated NMR Structure Calculation With CYANA.- NOE Assignment With ARIA 2.0.- Membrane Protein Structure Determination Using Solid-State NMR.