a peek into tropomyosin binding and unfolding on the actin filament一窥原肌球蛋白肌动蛋白丝绑定和展开.pdfVIP

A Peek into Tropomyosin Binding and Unfolding on the Actin Filament Abhishek Singh1,2,3¤*, Sarah E. Hitchcock-DeGregori1 1 Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America, 2 MD/PhD Program, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America, 3 Joint Graduate Program in Biochemistry and Molecular Biology, University of Medicine and Dentistry, New Jersey Graduate School of Biomedical Sciences and Rutgers University, Piscataway, New Jersey, United States of America Abstract Background: Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Principal Findings: Tropomyosin’s periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle a-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, a