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This volume explores current technologies used to investigate the formation, insertion, and function of metalloclusters associated with proteins. Chapters describe relevant topics about Fe-S cluster metabolism are explored through genetic, biochemical, spectroscopic methods. 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.
Authoritative and cutting-edge, Fe-S Proteins: Methods and Protocols aims to be a useful practical guide to researchers to help further their study in this field.
List of contents
Molecular biology and genetic tools to investigate functional redundancy among Fe-S cluster carriers in E. coli.- Study Fe-S cluster proteins in Methanococcus maripaludis, a model archaeal organism.- Genetic approaches to uncover gene products involved in iron-sulfur protein maturation: High throughput genomic screening using transposon-sequencing.- Methods for heterologous overproduction of Fe-S proteins.- Enzymatic and Chemical in vitro Reconstitution of Iron-Sulfur Cluster Proteins.- Expression, isolation, and characterization of vanadium nitrogenase from Azotobacter vinelandii.- Ni-NTA affinity chromatography to characterize protein-protein interactions during Fe-S cluster biogenesis.- tRNA modifications as a readout of S and Fe-S metabolism.- Methods to unravel the roles of ATPases in Fe-S cluster biosynthesis.- Methods to investigate the kinetic profile of cysteine desulfurases.- A fast and ratiometric method for quantification of cysteine-bound persulfides based on alkylation and gel-shift assays.- Circular dichroism to probe the synthesis, transfer, and stability of Fe-S clusters.- Native mass spectrometry of iron-sulfur proteins.- Characterization of Paramagnetic Iron-Sulfur Clusters using Electron Paramagnetic Resonance Spectroscopy.- Characterization of Fe-S clusters in proteins by Mössbauer Spectroscopy.- Biochemical approaches to probe the role of the auxiliary iron-sulfur cluster of lipoyl synthase from Mycobacterium tuberculosis.- Methods to Screen for Radical SAM Enzyme Crystallization Conditions.
