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Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation
Published on: September 29, 2019
Target binding to S100B reduces dynamic properties and increases Ca(2+)-binding affinity for wild type and EF-hand
Melissa A Liriano1, Kristen M Varney, Nathan T Wright
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
Journal of Molecular Biology
|July 25, 2012
Summary
Mutations in S100B
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- S100B is a calcium-binding protein involved in various cellular processes.
- Understanding S100B's calcium binding and dynamics is crucial for elucidating its function.
- TRTK-12 is a known target of S100B.
Purpose of the Study:
- To investigate the role of the second EF-hand in S100B's calcium binding and dynamic properties.
- To determine how binding to the target TRTK-12 affects S100B's calcium affinity and dynamics.
Main Methods:
- Site-directed mutagenesis to create EF2-hand mutants of S100B (D61N, D63N, D65N, E72A).
- Isothermal titration calorimetry (ITC) to measure calcium binding affinities.
- Nuclear Magnetic Resonance (NMR) spectroscopy, including relaxation dispersion, to assess protein dynamics.
- X-ray crystallography to determine structural changes upon calcium and TRTK-12 binding.
Main Results:
- EF2-hand mutations significantly reduced calcium binding affinity compared to wild-type S100B in the absence of TRTK-12.
- TRTK-12 binding increased the calcium-binding affinity of both wild-type and mutant S100B proteins.
- NMR studies revealed that TRTK-12 binding reduced protein dynamics (R(ex) and backbone motions).
- X-ray structures did not show altered calcium coordination, suggesting dynamics, not static structure, explain affinity changes.
Conclusions:
- The second EF-hand of S100B is critical for high-affinity calcium binding.
- Target binding by TRTK-12 enhances S100B's calcium-binding affinity through modulation of protein dynamics.
- A model is proposed where altered protein dynamics upon TRTK-12 binding allosterically increase calcium affinity.
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