Mechanism Of Actomyosin Dissociation Affected By Myosin Regulatory Light Chain Phosphorylation

It was found that phosphorylation of myosin regulatory light chain(MRLC)affected the structure of actomyosin cross-bridge in vivo,while actomyosin dissociation affected the tenderness and water holding capacity of meat during postmortem.However,MRLC phosphorylation in postmortem muscles affects actomyosin dissociation is worth exploring.The mechanism of actomyosin dissociation affected by MRLC phosphorylation was studied by SDS-PAGE,Pro-Q and Ruby fluorescence staining,Western Blot,phosphoproteomics,Discovery Studio software simulation,and Isothermal Titration Calorimetry(ITC)analysis.Firstly,the effect of MRLC phosphorylation on actomyosin dissociation and myosin degradation during postmortem was determined in situ model.Secondly,MLCK and ML-7 were used to regulate the MRLC phosphorylation level of muscle homogenate,the relationship between MRLC phosphorylation and actomyosin dissociation was verified in vitro.And then analyzing the phosphorylated peptides and sites of MRLC which was closely related to actomyosin dissociation by phosphoproteomics.Finally,Discovery Studio software simulation,ITC,and protein-protein interaction analysis were used to study the effect of MRLC phosphorylation at characteristic site on actomyosin dissociation.The results are as follows:(1)MRLC phosphorylation negatively affected actomyosin dissociation,as well as the degradation of MRLC and MHC.This study showed that the level of MRLC phosphorylation is negatively correlated with the degree of actomyosin dissociation(r=-0.794,P < 0.05),the degradation of MRLC(r=-0.764,P < 0.05)and MHC(r=-0.826,P < 0.05)during 0.5-72 h postmortem.(2)The in vitro model verified that MRLC phosphorylation negatively affected actomyosin dissociation,which was consistent with the results of in situ model.It is suggested that MRLC phosphorylation may contribute to the formation of interaction between myosin and actin,thereby inhibiting the dissociation of actomyosin.There was a significant negative correlation between MRLC phosphorylation and actomyosin dissociation(r =-0.736,P < 0.05).(3)Ser15 of MRLC was the most closely related phosphorylation site that affects actomyosin dissociation.MRLC phosphorylation status at Ser15 was negatively correlated with actomyosin dissociation in sheep muscles(r=-0.816,P < 0.05).The phosphorylation of some muscle contraction proteins,regulatory proteins,and structural proteins,particularly MRLC,MHC,titin,and nebulin,was associated with actomyosin dissociation.(4)There are two possible pathways how MRLC phosphorylation at Ser15(Ser17)affects actomyosin dissociation.Firstly,MRLC phosphorylation at Ser15 inhibited actomyosin dissociation through increasing the interaction of proteins,such as ionic bonds,hydrogen bonds and hydrophobic interactions.Secondly,MRLC phosphorylation at Ser15 decreases the kinetic energy and total energy of actomyosin,thereby inhibited actomyosin dissociation.Amino acid sequence matching of MRLC revealed that the Ser15 in sheep skeletal muscle corresponds to the Ser17 in rabbit skeletal muscle.