1. Direct Observation of Ca2+-Induced Calmodulin Conformational Transitions in IntactXenopus laevisOocytes by19F NMR Spectroscopy
- Author
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Yansheng Ye, Xiaoli Liu, Maili Liu, Guohua Xu, and Conggang Li
- Subjects
Models, Molecular ,Myosin light-chain kinase ,Calmodulin ,Protein Conformation ,Population ,Xenopus ,Catalysis ,Xenopus laevis ,Protein structure ,Isotopes ,Myosin ,Animals ,education ,Myosin-Light-Chain Kinase ,Nuclear Magnetic Resonance, Biomolecular ,education.field_of_study ,biology ,Chemistry ,General Medicine ,Fluorine ,General Chemistry ,Nuclear magnetic resonance spectroscopy ,biology.organism_classification ,Biochemistry ,Oocytes ,Biophysics ,biology.protein ,Calcium ,Target protein ,Protein Binding - Abstract
The Ca(2+) -mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by (19) F NMR spectroscopy of the transition of the Ca(2+) -free and -bound forms in Xenopus laevis oocytes at different Ca(2+) levels. We find that the Ca(2+) -bound CaM population increased greatly upon binding the target protein myosin light-chain kinase (MLCK) at the same Ca(2+) level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long-range structural constraints in cells. Our study shows that (19) F NMR spectroscopy can be used to obtain long-range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants.
- Published
- 2015
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