Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: Application to myosin light chain 2 |
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Authors: | Andrew J Dingley Joel P Mackay Bogdan E Chapman Michael B Morris Philip W Kuchel Brett D Hambly Glenn F King |
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Institution: | (1) Department of Biochemistry, University of Sydney, 2006 Sydney, New South Wales, Australia;(2) Department of Pathology, University of Sydney, 2006 Sydney, New South Wales, Australia |
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Abstract: | Summary At the millimolar concentrations required for structural studies, NMR spectra of the calcium-binding protein myosin light chain 2 (MLC2) showed resonance line widths indicative of extensive self-association. Pulsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt derivative 3-(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). PFG NMR measurements indicated that CHAPS was capable of preventing MLC2 self-association, but only at concentrations well above the critical micelle concentration of 7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 correponded to a protein monomer plus seven molecules of bound detergent. The resolution and sensitivity of 2D 15N-1H HSQC spectra of MLC2 were markedly improved by the addition of 25 mM CHAPS, consistent with a reduction in aggregation following addition of the detergent. The average amide nitrogen T2 value for MLC2 increased from 30 ms in the absence of CHAPS to 56 ms in the presence of 25 mM CHAPS. The results of this study lead us to propose that PFG NMR spectroscopy can be used as a facile alternative to conventional techniques such as analytical ultracentrifugation for examining the self-association of biological macromolecules. |
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Keywords: | Pulsed-field-gradient NMR Translational diffusion coefficient Self-association Myosin light chain CHAPS |
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