Fe-S Proteins, 1st ed. 2021
Methods and Protocols
Methods in Molecular Biology Series, Vol. 2353

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.

Part I - Methods for genetic manipulation and Fe-S protein expression

1. Molecular biology and genetic tools to investigate functional redundancy among Fe-S cluster carriers in E. coli

Yohann Duverger and Béatrice Py

2. Study Fe-S cluster proteins in Methanococcus maripaludis, a model archaeal organism

Cuiping Zhao, Christina A Roberts, Ian J Drake, and Yuchen Liu

3. Genetic approaches to uncover gene products involved in iron-sulfur protein maturation: High throughput genomic screening using transposon-sequencing

Valerie J. Carabetta, Karla Esquilin-Lebron, Ehud Zelzion, Jeffrey M. Boyd

4. Methods for heterologous overproduction of Fe-S proteins

Elliot I. Corless and Edwin Antony

5. Enzymatic and Chemical in vitro Reconstitution of Iron-Sulfur Cluster Proteins

Mauro Marengo, Rita Puglisi, Simonetta Oliaro-Bosso, Annalisa Pastore and Salvatore Adinolfi

6. Expression, isolation, and characterization of vanadium nitrogenase from Azotobacter vinelandii

Katharina Grunau, Jakob Gies-Elterlein, Christian Trncik and Oliver Einsle

Part II - Biochemical methods to probe steps involved in the synthesis of Fe-S clusters

7.Ni-NTA affinity chromatography to characterize protein-protein interactions during Fe-S cluster biogenesis

Terrell D. Carter and F. Wayne Outten

8.  tRNA modifications as a readout of S and Fe-S metabolism

Ashley M. Edwards, Maame A. Addo, Patricia C. Dos Santos

9. Methods to unravel the roles of ATPases in Fe–S cluster biosynthesis

Christa N. Molé, Kinjal Dave, Deborah L. Perlstein

10. Methods to investigate the kinetic profile of cysteine desulfurases

Maame A. Addo, Ashley M. Edwards, Patricia C. Dos Santos

11. A fast and ratiometric method for quantification of cysteine-bound persulfides based on alkylation and gel-shift assays

Sylvain Gervason, Batoul Srour, and Benoit D’Autréaux

Part III - Biophysical methods to study Fe-S clusters and reactions promoted by metalloenzymes

12. Circular dichroism to probe the synthesis, transfer, and stability of Fe-S clusters

Stefania Iametti, Francesco Bonomi, and Alberto Barbiroli

13. Native mass spectrometry of iron-sulfur proteins

Jason C. Crack, and Nick E. Le Brun

14. Characterization of Paramagnetic Iron-Sulfur Clusters using Electron Paramagnetic Resonance Spectroscopy

Troy A. Stich

15. Characterization of Fe-S clusters in proteins by Mӧssbauer Spectroscopy

Chie Ueda, Michelle Langton, and Maria-Eirini Pandelia

16. Biochemical approaches to probe the role of the auxiliary iron-sulfur cluster of lipoyl synthase from Mycobacterium tuberculosis

Vivian Robert Jeyachandran,Jay V. Pendyala, Erin L. McCarthy, Amie K. Boal, and Squire J. Booker

17. Methods to Screen for Radical SAM Enzyme Crystallization Conditions

Lydie Martin, Xavier Vernède, and Yvain Nicolet

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts