PROJECT SUMMARYArthrogryposis is present in 1 in 3000 live births causing joint contractures in both upper and lower limbs.There is no cure making it an unmet medical need. Mutations in the MYBPC1 gene encoding slow skeletalmyosin-binding protein C (sMyBP-C) expressed in both slow and fast muscle types are associated withdistal arthrogryposis (DA). MYBPC2 encodes for fast skeletal MyBP-C (fMyBP-C) and is found only in fast-twitch muscle. As a myosin-anchored protein of muscle MyBP-C extends toward actin positioned centrally inthe sarcomere to regulate actomyosin interactions in force development. MyBP-C in skeletal muscle has threemajor regulators: isoform (slow vs. fast) splice variant (long vs. short sMyBP-C) and posttranslationalmodification (phosphorylation). sMyBP-C is phosphorylated by protein kinase A (PKA) at its N terminus. Therole(s) of sMyBP-C its phosphorylation and DA mutations in skeletal muscle are not known. Our preliminarystudies of sMyBP-C show that binding to actomyosin is dependent on phosphorylation and DA mutations. Wehave developed innovative biophysical tools that enable evaluation of skeletal MyBP-C structural dynamicsactomyosin interactions in muscle and effects of phosphorylation and mutations. Our new preliminary studiesdemonstrate that we have successfully developed fluorescent sensors in N terminal sMyBP-C whose structureand dynamics are sensitive to PKA-mediated phosphorylation and binding to actin. We have also developedinter-molecular fluorescence assays that resolve actin binding between fMyBP-C long sMyBP-C and shortMyBP-C due to phosphorylation and the presence of tropomyosin on actin. These preliminary results suggestkey physiological mechanisms of regulation for the different skeletal MyBP-C and provides additional scientificpremise and feasibility for pursuing the proposed studies. Aim 1 will evaluate effects of sMyBP-C binding andDA mutations on interactions with actomyosin capturing structure and proximities of N terminal sMyBP-Cactin and myosin. Spectroscopic probes will be placed in these proteins and approaches will be employed todetect key conformations in vitro and in situ with wild type and DA mutant sMyBP-C. For fiber experimentsmuscle will be isolated from novel sMyBP-C knockout (KO) mice and permeabilized with recombinant sMyBP-C DA mutants and muscle protein probes. Samples will be assessed for binding and contractile function. Aim2 will determine how PKA-mediated phosphorylation of sMyBP-C affects the parameters evaluated in Aim 1.Aim 3 will determine how fMyBP-C affects the parameters evaluated in Aim 1 except using fMyBP-C KO andsMyBP-C/fMyBP-C double-KO mice for fibers experiments. The proposed studies capture structural dynamicsand interactions in real time and myofilament space using novel high-resolution approaches. These aimsoutline a stepwise plan for studying normal and mutant skeletal MyBP-C during the contractile cycle. Bymonitoring distances between points on proteins and the order (or disorder) of those distances underphysiological conditions mutants can be separated into bins to facilitate targeted mechanistic-based therapies.