Structural Studies of Interactions between Cardiac Troponin I and Actin in Regulated Thin Filament Using Forster Resonance Energy Transfer

The Ca2+-induced interaction between cardiac troponin I (cTnl) and actin plays a key role in the regulation of cardiac muscle contraction and relaxation. In this report we have investigated changes of this interaction in response to strong cross-bridge formation between myosin S1 and actin and PKA phosphorylation of cTnI within reconstituted thin filament. The interaction was monitored by measuring Forster resonance energy transfer (FRET) between the fluorescent donor 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (AEDANS) attached to the residues 131, 151, 160 167, 188, and 2 10 of cTnI and the nonfluorescent acceptor 4-(dimethylamino)phenylazophenyl-4'-maleimide (DABM) attached to cysteine 374 of actin. The FRET distance measurements showed that bound Ca2+ induced large increases in the distances from actin to the cTnI sites, indicating a Ca2+-triggered separation of cTnI from actin. Strongly bound myosin S1 induced additional increases in these distances in the presence of bound Ca2+. The two ligand-induced increases were independent of each other. These two-step changes in distances provide a direct link of structural changes at thin interface between cTnI and actin to the three-state model of thin filament regulation of muscle contraction and relaxation. When cTnC was inactivated through mutations of key residues within the 12-residue Ca2+-binding loop, strongly bound S1 alone induced increases in the distances in spite of the fact that the filaments no longer bound regulatory Ca2+. These results suggest bound Ca2+ or strongly bound S1 alone can partially activate thin filament, but full activation requires both bound Ca2+ and strongly bound S1. The distributions of the FRET distances revealed different structural dynamics associated with different regions of cTnI in different biochemical states. The second actin-binding region appears more rigid than the inhibitory/regulatory region. In the Mg2+ state, the regulatory region appears more flexible than the inhibitory region, and in the Ca2+ state the inhibitory region becomes more flexible. PKA phosphorylation of cTnI at Ser23 and Ser24 distance from actin to cTnI residue 131 by 2.2-5.2 A in different biochemical states and narrowed the distributions of the distances from actin to the inhibitory and regulatory regions of cTnI. The observed phosphorylation effects are likely due to an intramolecular interaction of the phosphorylated N-terminal segment and the inhibitory region of cTnI.