PHOSPHORYLATION OF REGULATORY LIGHT CHAIN AFFECTS SPATIAL ORIENTATION OF MYOSIN CROSS-BRIDGES IN RABBIT SKELETAL MUSCLE

Abstract

Purpose: Muscle is organized into regular periodic thick myosin and thin actin filaments. Myosin tails interact with each other to form a tight coiled coil rod, and the heads protrude out to interact with actin. Myosin head referred to as cross-bridge has ATPase activity and actin binding domain. The tail has site (Regulatory Light Chain (RLC) domain) which gets phosphorylated during muscle contraction. The degree to which RLC phosphorylation affects the organization of myosin cross-bridges is the question investigated in this project. Methods: Rabbit psoas muscle is the source of sample for experiments in this project. Glycerinated muscle bundles were homogenized and myofibrils were extracted. Myofibrils were phosphorylated with skeletal myosin light chain kinase/calmodulin and dephosphorylated with phosphatase and 1mM EGTA. Essential light chain (LC1) protein site specifically labeled with SeTau dye was exchanged with native LC1 at a minimal concentration in myofibrils. Labeled myofibrils were analyzed for error of the mean of polarized fluorescence (which is a measure of orientation) emanating from myosin cross-bridges. Results: Histograms were plotted from the polarized fluorescence data and the Full Width at Half Maximum (FWHM) of the mean was calculated. This is an index of cross-bridge order. FWHM values in relaxed de-phosphorylated and phosphorylated muscle were 0.213 ± 0.015, and 0.23 ± 0.022, respectively. During active contraction FWHM values in de-phosphorylated and phosphorylated muscle were 0.23 ± 0.039 and 0.387 ± 0.050, respectively. In 20 experiments, statistically significant difference was seen between the groups. Conclusions: Phosphorylation of the RLC disturbs myosin head-to-head and head-to-rod interactions. Low FWHM in de-phosphorylated muscle suggests well organized cross-bridges. Phosphorylation destroys this organization, as indicated by high FWHM.