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) sho wed resonance line widths indicative of extensive self-association. Pu lsed-field-gradient (PFG) NMR spectroscopy was used to examine whether MLC2 aggregation could be prevented by the zwitterionic bile salt der ivative 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate (CH APS). PFG NMR measurements indicated that CHAPS was capable of prevent ing MLC2 self-association, but only at concentrations well above the c ritical micelle concentration of similar to 7.5 mM. CHAPS was most eff ective at a concentration of 22.5 mM, where the apparent molecular mas s of MLC2 corresponded to a protein monomer plus seven molecules of bo und detergent. The resolution and sensitivity of 2D N-15-H-1 HSQC spec tra of MLC2 were markedly improved by the addition of 25 mM CHAPS, con sistent with a reduction in aggregation following addition of the dete rgent. The average amide nitrogen T-2 value for MLC2 increased from si milar to 30 ms in the absence of CHAPS to similar to 56 ms in the pres ence 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 conventio nal techniques such as analytical ultracentrifugation for examining th e self-association of biological macromolecules.