This volume summarizes the state-of-the-art methodology for the functional characterization of phytochromes in vitro and in vivo. Chapters and divided into four parts covering, phytochrome discovery, spectroscopic techniques, x-ray crystallography, x-ray crystallography, and analyzing phytochrome function in vitro and in vivo. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and comprehensive, Phytochromes: Methods and Protocols aims to be a versatile laboratory bench and computational resource that is an essential reference.
Discovery and Engineering of Unique Cyanobacteriochrome GAF Domains.- Analysis of Sequence Diversity in Sub-families of Phytochrome-linked Effectors.- Strategies for Co-expression of Bilin-producing Enzymes in Escherichia coli.- Preparation of Phycocyanobilin (PCB) with and without 13C/15N Isotope Labelling for in vitro Phytochrome Autoassembly.- Production of Plant Phytochromes by Combining Insect Cells and Bacterial Expression Systems.- Overcoming the challenges of producing plant phytochromes in Escherichia coli.- Characterizing Phytochrome Photochemistry Using UV-Visible Spectroscopy in Host Cell Extracts and Purified Protein.- Computational Protocols to Study Phytochromes.- Using Computational Techniques for the Characterization of Structural Changes During the Phytochrome Photocycle.- FTIR Spectroscopy of Red and Far-red Light Sensing Phytochromes in vitro.- Spectroscopic Analyses of Photoconversion in Phytochromes.- Small-angle X-ray Scattering Studies on Phytochromes.- A Simple Modification of a Commercial Plunge-freezer to Allow Time-resolved Vitrification for Cryo-EM Analyses of Light-activated Proteins.- Structures of Myxobacterial Phytochrome Revealed by Cryo-EM.- Structural Analysis of Phytochrome Photoreceptors using Hydrogen-Deuterium Exchange Coupled to Mass Spectrometry.- Encapsulation of Phytochrome Proteins in Trehalose Matrices: Practical Considerations for the Use in Solid-State NMR Spectroscopy.- Analyzing in vitro Autokinase Activity of Microbial Phytochromes under well-defined Light Conditions.- Detection of Histidine Kinase activities of Bacteriophytochromes with Phos-tag™ Acrylamide Gel Electrophoresis.- Assessing the Role of PPKs in Phosphorylating Phytochrome A using the Protoplast Transient Expression System.- A Fast and Reliable Nuclei Extraction Method for Phytochrome Functional Analysis.- Opto-APCs: Displaying Phytochrome B on the Surface of Living Cells for Optogenetic Studies.- Coupling Recombinant PhyB to Cell Culture Plates and Beads.
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