A Disulfide Bond at Cys 190 of Tropomyosin Alters Tryptic Cleavage Pattern

Abstract

Native tropomyosin (Tm), an α-helical coiled-coil, possesses a charged acidic amino acid (Asp 137) that occurs in a hydrophobic position which destabilizes the coiled-coil. This region is sensitive to tryptic cleavage and is important in the proper regulation of the myosin activate ATPase, (Sumida, John P., Wu, Eleanor, Lehrer, Sherwin S, JBC 283, 2008). Thermal stability measurements of Tm suggest a long-range interaction between the Asp 137 position and the Cys 190 position. In the current work, we present further evidence of long-range interactions along the length of tropomyosin.Native frog Tm was cross-linked with DTNB at Cys 190. Tryptic cleavage patterns of this cross-linked species were compared to a control Tm in which the Cys 190 groups were maintained reduced state using DTT. The course of tryptic cleavage of the cross-linked molecule was altered compared to the fully reduced molecule. Proteolysis of the cross-linked molecule produced new tryptic fragments seen in a reduced SDS gel at 30kDa and 20kDa. Trypsin cleaves reduced tropomyosin at Arg 133 to produce two new tryptic fragments (17kDa and 15kDa). The observation that cross-linked Tm exhibits a change in the cleavage rate, and affects the cleavage pattern, demonstrates a long-range effect of the cross-link at Cys 190 on the site of trypsin cleavage, Arg 133.This finding adds evidence to the idea that “flexibility” is associated with a locally fluctuating region in the middle of the molecule which has implications on our understanding of how Tm moves on the actin surface, and provides insight into understanding how single point mutations in Tm may result in dysfunction and disease associated with various myopathies.