Vol. 93, No. 1,1980 March 13, 1980 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 209-214 LIGHT CHAIN PHOSPHORYLATION ALTERS THE CONFORMATION OF SKELETAL MUSCLE MYOSIN Carolyn J. Ritz-Gold*, Roger Cooke*, Donald K. Blumenthal#, and James T. Stull# * Cardiovascular Research Institute and the Department of Biochemistry and Biophysics, University of California, San Francisco 94143 and #Moss Heart Center and Department of Pharmacology University of Texas Health Sciences Center at Dallas Dallas, Texas 75235 Received January 18, 1980 SUMMARY: Using limited proteolysis by chymotrypsin or papain, we examined the effects of phosphorylation on the conformation of skeletal muscle myosin. In the absence of MgCI2, phosphorylation of the 19,000 dalton light chain (LC2) inhibited digestion of LC 2 by chymotrypsin or papain. Phosphorylation also suppressed chymotryptic conversion of the heavy chain to suhfragment i and increased its conversion to heavy meromyosin. These results indicate that phosphorylation alters the conformation of the N-terminal segment of LC 2 and suggest that it also affects the heavy chain. In the presence of MgCI9, phosphorylation inhibited the chymotryptic digestion of LC 2 but an effect on digestion of the heavy chain was not apparent. Thus, phosphorylation of LC 2 alters LC 2 conformation under physiological conditions. INTRODUCTION When skeletal muscle contracts, the 19,000 dalton light chain of myosin is phosphorylated by calcium-dependent light chain kinase (1-3). In rat skeletal muscle, the extent of phosphorylation was found to correlate with the degree of post-tetanic potentiation of isometric twitches (4). These findings suggest that phosphorylation might participate in regulating skeletal myosin. Since regulation by phosphorylation is known to be mediated by conformational changes in other pro- teins (5,6), conformational changes may also mediate a regulatory effect of phos- phorylation in skeletal muscle. However, the effects of phosphorylation on the conformation of intact skeletal myosin have not been defined. Since limited proteolysis is a sensitive tool for probing protein conforma- tional changes (7), we investigated the effects of phosphorylation on myosin conformation by looking at changes in susceptibility to digestion at four sites in myosin that are especially sensitive to chymotryptle cleavage. Three of these sites are cleaved sequentially: cleavage at phe 18 within the N-terminal segment of LC 2 permits cleavage at a second site in LC 2 (phe 53) (8,9); this in turn permits degradation of LC 2 and cleavage of the myosin heavy chain at the flexible "swivel" site (i0) joining the head to the rod, producing free heads [subfragment Abbreviations used: LC2, the 19,000 dalton light chain of skeletal muscle myosin; LC2-P , the phosphorylated form of LC 2. 0006-29]X/80/050209-06501.00/0 Copyright © 1980byAcadem~ Press, Inc. 209 All righ~ofreproduction m anyform reserved.