Structural and functional characterization of calponin fragments

Area of Science:

• Biochemistry
• Molecular Biology
• Muscle Physiology

Background:

• Calponin is a protein found in smooth muscle.
• Its role in muscle contraction is not fully understood.
• Calponin is known to interact with actin and myosin.

Purpose of the Study:

• To investigate the functional domains of chicken gizzard calponin.
• To determine the effect of phosphorylation on calponin's activity.
• To identify the sites of phosphorylation in calponin.

Main Methods:

• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing to separate calponin components.
• Limited proteolysis with 2-nitro-5-thiocyanobenzoic acid to generate fragments.
• Biochemical assays to assess actin-, tropomyosin-, and calmodulin-binding.
• Enzyme activity assays to measure inhibition of MgATPase activity.
• Phosphorylation studies using protein kinase C and subsequent tryptic digestion.

Main Results:

• Chicken gizzard calponin consists of two separable components, likely isoforms.
• Purified 30 kDa and 21 kDa fragments retained actin-, tropomyosin-, and calmodulin-binding properties.
• Both fragments inhibited the actin-activated MgATPase activity of smooth muscle myosin.
• Protein kinase C phosphorylation of calponin fragments inhibited their actin binding and ATPase inhibitory activity.
• Tryptic digestion of phosphorylated calponin yielded three phosphopeptides with distinct N-terminal sequences, each containing a single phosphoserine.

Conclusions:

• Calponin's functional domains responsible for actin binding and myosin ATPase inhibition are located within the 30 kDa and 21 kDa fragments.
• Phosphorylation by protein kinase C modulates calponin's interaction with actin and its inhibitory function.
• Phosphorylation occurs on serine residues within specific N-terminal regions of the calponin molecule.