Measuring protein self-association using pulsed-field-gradient NMR spectroscopy: application to myosin light chain 2.

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 approximately 7.5 mM. CHAPS was most effective at a concentration of 22.5 mM, where the apparent molecular mass of MLC2 corresponded 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 approximately 30 ms in the absence of CHAPS to approximately 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.