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Informationen zum Autor 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. Klappentext Nuclear Magnetic Resonance (NMR) spectroscopy, a physical phenomenon based upon the magnetic properties of certain atomic nuclei, has found a wide range of applications in life sciences over recent decades. The dramatic advances in NMR techniques have led to corresponding advances in the ability of NMR to study structure, dynamics and interactions of biological macromolecules in solution under close to physiological conditions. This volume focuses on the use of NMR to study proteins. NMR can be used to determine detailed three-dimensional structures of proteins in solution. Furthermore, it provides information about conformational or chemical exchange, internal mobility and dynamics at timescales varying from pcoseconds to seconds. It is the primary technique used to obtain information on intrinsically disordered (unfolded) proteins, since these proteins will not crystallize easily. NMR is also a very powerful method for the study of interactions of protein with other molecules, whether small molecules (including drugs), nuclear acids or other proteins. This up-to-date volume covers NMR techiniques and their application to proteins, with a focus on practical details. This book will provide a newcomer to NMR with the practical guidance in order to carry out successful experiments with proteins and to analyze the resulting spectra. Those who are familiar with the chemical applications of NMR will also find is useful in understanding the special requirements of protien NMR. Zusammenfassung Nuclear Magnetic Resonance (NMR) spectroscopy, a physical phenomenon based upon the magnetic properties of certain atomic nuclei, has found a wide range of applications in life sciences over recent decades. This up-to-date volume covers NMR techniques and their application to proteins, with a focus on practical details. Inhaltsverzeichnis List of Contributors xiii Introduction 1 Lu-Yun Lian and Gordon Roberts References 4 1 Sample Preparation, Data Collection and Processing 5 Frederick W. Muskett 1.1 Introduction 5 1.2 Sample Preparation 5 1.2.1 Initial Considerations 6 1.2.2 Additives 7 1.2.3 Sample Conditions 7 1.2.4 Special Cases 8 1.2.5 NMR Sample Tubes 9 1.2.5.1 3 mm Tubes 10 1.3 Data Collection 11 1.3.1 Locking 11 1.3.2 Tuning 11 1.3.3 Shimming 12 1.3.4 Calibrating Pulses 13 1.3.5 Acquisition Parameters 14 1.3.6 Fast Acquisition Methods 16 1.4 Data Processing 17 References 20 2 Isotope Labelling 23 Mitsuhiro Takeda and Masatsune Kainosho 2.1 Introduction 23 2.2 Production Methods for Isotopically Labelled Proteins 24 2.2.1 Recombinant Protein Expression in Living Organisms 24 2.2.1.1 Escherichia coli 24 2.2.1.2 Yeast Cells 25 2.2.1.3 Other Host Cells 25 2.2.2 Cell-Free Synthesis 25 Protocol 1: Preparation of the Amino Acid Free S30 Extract 26 Protocol 2: Cell-Free Reaction on a Small Scale 28 2.3 Uniform Isotope Labelling of Proteins 29 2.3.1 Uniform 15N Labelling 29 2.3.2 Uniform 13C, 15N Labelling 30 2.3.3 2H Labelling 30 2.4 Selective Isotope Labelling of Proteins 32 2.4.1 Amino Acid Type-Selective Labelling 32 2.4.2 Reverse Labelling 34 2.4.3 Stereo-Selective Labelling 36 2.5 Segmental Labelling 37 2.6 SAIL Methods 38 2.6.1 Concept of SAIL 38 2.6.2 Practical Procedure for the SAIL Method 41 Protocol 3: Production of SAIL Proteins by the E. coli Cell-Free Method 41 2.6.3 Residue-Selective...
