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Protein conformational changes studied by diffusion NMR spectroscopy: application to helix-loop-helix calcium binding
Aalim M Weljie1, Aaron P Yamniuk, Hidenori Yoshino
1Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Alberta, Canada.
Protein Science : a Publication of the Protein Society
|January 23, 2003
Summary
Pulsed-field gradient diffusion NMR spectroscopy effectively measures conformational changes in calcium-binding proteins. This technique accurately screens protein-ligand and protein-protein interactions, revealing molecular size changes upon calcium binding or target interaction.
Area of Science:
- Biochemistry and Biophysics
- Structural Biology
- Nuclear Magnetic Resonance Spectroscopy
Background:
- Calcium-binding proteins play crucial roles in cellular signaling and regulation.
- Understanding their conformational dynamics upon calcium saturation and target binding is essential for elucidating their function.
- Helix-loop-helix regulatory proteins are a significant class of calcium-binding proteins.
Purpose of the Study:
- To characterize conformational changes in helix-loop-helix regulatory calcium-binding proteins using pulsed-field gradient (PFG) diffusion NMR spectroscopy.
- To evaluate the utility of PFG-diffusion NMR for screening molecular size changes in protein-ligand and protein-protein interactions.
- To compare PFG-diffusion NMR results with existing data, such as small-angle X-ray scattering.
Main Methods:
- Pulsed-field gradient (PFG) diffusion NMR spectroscopy was employed to determine hydrodynamic radii (R(h)).
- Calmodulin (CaM) served as a model system for evaluating the technique.
- The method was applied to various CaM-ligand complexes and other calcium-binding proteins, including CIB, calbrain, and CDPK.
Main Results:
- PFG-diffusion NMR accurately determined hydrodynamic radii (R(h)) for apo- and Ca(2+)-calmodulin, consistent with prior studies.
- Binding of CaM to target peptides (MLCK, PDE, SIV) induced a collapse, confirmed by comparison with small-angle X-ray scattering data.
- CaM-cisplatin complex showed an unexpected near-complete collapse, while CaMKI and SIV-N peptides induced varying degrees of collapse.
- Calcium binding to CIB, calbrain, and CDPK resulted in decreased R(h), attributed to shifts from unfolded to folded conformations, with calbrain forming a dimer.
Conclusions:
- PFG-diffusion NMR is a rapid and accurate method for assessing molecular size changes in calcium-binding proteins.
- The technique is effective for screening protein-ligand and protein-protein interactions, providing insights into conformational dynamics.
- Conformational changes, including collapse and folding, are prevalent upon calcium binding and target interaction in this protein class.