Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding tag
| Autor: | Philippe S. Nadaud, Sunil Saxena, Rajith J. Arachchige, Timothy F. Cunningham, Min Gao, Christopher P. Jaroniec, Charles D. Schwieters, Ishita Sengupta |
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| Rok vydání: | 2014 |
| Předmět: |
biology
Mutation Missense Nuclear magnetic resonance spectroscopy Cyclams Biochemistry Article Protein Structure Tertiary Paramagnetism chemistry.chemical_compound Crystallography Protein structure Solid-state nuclear magnetic resonance Cyclen chemistry Amino Acid Substitution Bacterial Proteins Heterocyclic Compounds Magic angle spinning biology.protein Protein G Spectroscopy Nuclear Magnetic Resonance Biomolecular Copper |
| Zdroj: | Journal of biomolecular NMR. 61(1) |
| ISSN: | 1573-5001 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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