title: Cu²⁺ as an ESR Probe of Protein Structure and Function.
abstract: Electron spin resonance (ESR) spectroscopy in combination with site-directed spin labeling has been widely used to determine the structure and dynamics of proteins and other biomolecules. The most popular spin label is a nitroxide-based radical which can be attached to a protein via a site-specific reaction with either native cysteines or cysteines engineered into the system via site-directed mutagenesis. Paramagnetic transition metals, including Cu(2+), often serve as cofactors of metalloproteins, and have already been realized as ESR probes to report structural information in these proteins. This chapter summarizes recent methodological development from our laboratory in utilizing Cu(2+) as an ESR spin probe to determine distance information. We focus on detailed experimental procedures, optimized instrumental parameters, and data analysis approaches in order to guide one who is new to the field. Theory and applications of metal ESR have been reviewed in literature and are not the focus of this chapter. A few examples of applications of the methods are listed in the end.
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Zhongyu
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Ji
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Cunningham
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. Electronic address: sksaxena@pitt.edu.
authors.forename: Sunil
authors.initials: S
authors.lastname: Saxena
mesh_terms: D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D009589:Nitrogen Oxides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D013113:Spin Labels; D013329:Structure-Activity Relationship
chemical_list: D009589:Nitrogen Oxides; D011506:Proteins; D013113:Spin Labels; C039900:nitroxyl; D003545:Cysteine
keywords: Copper; Distance measurements; Electron spin resonance spectroscopy; Protein structure