title: Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag.
abstract: Paramagnetic relaxation enhancements (PREs) are a rich source of structural information in protein solid-state NMR spectroscopy. Here we demonstrate that PRE measurements in natively diamagnetic proteins are facilitated by a thiol-reactive compact, cyclen-based, high-affinity Cu(2+) binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), that overcomes the key shortcomings associated with the use of larger, more flexible metal-binding tags. Using the TETAC-Cu(2+) K28C mutant of B1 immunoglobulin-binding domain of protein G as a model, we find that amino acid residues located within ~10 Å of the Cu(2+) center experience considerable transverse PREs leading to severely attenuated resonances in 2D (15)N-(13)C correlation spectra. For more distant residues, electron-nucleus distances are accessible via quantitative measurements of longitudinal PREs, and we demonstrate such measurements for (15)N-Cu(2+) distances up to ~20 Å.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210, USA.
authors.forename: Ishita
authors.initials: I
authors.lastname: Sengupta
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.forename: Rajith J
authors.initials: RJ
authors.lastname: Arachchige
authors.forename: Philippe S
authors.initials: PS
authors.lastname: Nadaud
authors.forename: Timothy F
authors.initials: TF
authors.lastname: Cunningham
authors.forename: Sunil
authors.initials: S
authors.lastname: Saxena
authors.forename: Charles D
authors.initials: CD
authors.lastname: Schwieters
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Jaroniec
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry*; D000080027:Cyclams; D006571:Heterocyclic Compounds / Q000737:chemistry*; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D000080027:Cyclams; D006571:Heterocyclic Compounds; C034284:IgG Fc-binding protein, Streptococcus; D003300:Copper; C038072:cyclen