A GENETICALLY-ENGINEERED, PROTEIN-BASED OPTICAL BIOSENSOR OF MYOSIN-II REGULATORY LIGHT-CHAIN PHOSPHORYLATION

Myosin II is an important motor in the contraction of smooth and stria ted muscle as well as in a variety of nonmuscle cell motile events inc luding cytokinesis, cortical contractions during migration of fibrobla sts, and capping of receptors. Phosphorylation of the 20-kDa light cha in by myosin light chain kinase is part of the regulation of smooth mu scle and mammalian nonmuscle myosin II. We designed, characterized, an d tested the use of a protein-based optical biosensor to monitor this phosphorylation ''switch.'' A regulatory light chain was genetically e ngineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys(18).LC(20)), reacted with the fluorophore acry lodan, responded to phosphorylation of serine 19 with a fluorescence e mission quenching of 60% and a 28-nm red-shift. When the acrylodan-lab eled mutant light chain (AC-Cys(18).LC(20)) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phos phorylation, K(+)ATPase activity, and in vitro motility. The acrylodan -labeled light chain was exchanged into the A-bands of chicken pectora lis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrate d that AC-Cys(18). LC(20) exchanged myofibrils expressed a phosphoryla tion-dependent fluorescence change. Labeled light chains were also inc orporated into stress fibers of living fibroblasts and smooth muscle c ells. This general approach of combining molecular biology and fluores cence spectroscopy to create novel protein-based optical biosensors sh ould provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temp oral-spatial chemical and molecular changes in live cells.