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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
Published on: September 17, 2017
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Protein structural studies by paramagnetic solid-state NMR spectroscopy aided by a compact cyclen-type Cu(II) binding
Ishita Sengupta1, Min Gao, Rajith J Arachchige
1Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, 43210, USA.
Journal of Biomolecular NMR
|November 30, 2014
Summary
We developed a new copper-binding tag (TETAC) to improve paramagnetic relaxation enhancements (PREs) in protein solid-state NMR. This method enhances structural insights for diamagnetic proteins up to 20 Å.
Area of Science:
- Biochemistry
- Biophysics
- Structural Biology
Background:
- Paramagnetic relaxation enhancements (PREs) are crucial for determining protein structures using solid-state NMR.
- Existing methods often use large, flexible tags, posing challenges for PRE measurements in diamagnetic proteins.
Purpose of the Study:
- To introduce a compact, high-affinity copper(II) (Cu(2+)) binding tag, 1-[2-(pyridin-2-yldisulfanyl)ethyl]-1,4,7,10-tetraazacyclododecane (TETAC), to facilitate PRE measurements.
- To overcome limitations of current metal-binding tags in protein NMR spectroscopy.
Main Methods:
- Utilized a thiol-reactive, cyclen-based Cu(2+) binding tag (TETAC).
- Applied the TETAC-Cu(2+) tag to a K28C mutant of the B1 immunoglobulin-binding domain of protein G.
- Performed 2D (15)N-(13)C correlation NMR experiments to measure transverse and longitudinal PREs.
Main Results:
- Residues within ~10 Å of the Cu(2+) center showed significant transverse PREs, causing attenuated resonances.
- Quantitative longitudinal PRE measurements enabled determination of (15)N-Cu(2+) distances up to ~20 Å.
- The compact TETAC tag proved effective in enhancing PRE measurements.
Conclusions:
- The TETAC-Cu(2+) tag significantly advances PRE applications in solid-state NMR for structural biology.
- This method provides a robust approach for probing protein structures, especially for natively diamagnetic proteins.
- The study demonstrates the utility of compact, high-affinity tags for detailed structural analysis.
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