Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation

► Sf9 insect cell expressed and purified αTm is N-terminal acetylated. ► Pseudo-phosphorylated αTm S283D regulates filaments like phosphorylated muscle αTm. ► αTm S283D does not alter steady-state Ca2+ binding to the thin filament. ► αTm S283D slows filament Ca2+ dissociation and prolongs myofibril force relaxation. ► Tm phosphorylation contributes to the modulation of muscle relaxation.