Structural Instability of Tropomyosin FHC Mutants D175N and E180G Probed by Limited Trypsin Cleavage

Abstract

Physiological and biochemical studies have shown that intact and reconstituted muscle thin filaments containing FHC mutants of tropomyosin Tm-D175N and Tm-E180G have a greater Ca2+-sensitivity than native systems. These mutant molecules have a lower thermal stability and bind less strongly to actin (Golitsina et al., 1997; Kremneva et al., 2004). Here we report trypsin digestion kinetics and cleavage sites using PAGE-SDS gels, MALDI MS and N-terminal sequencing. Native Tm is initially cleaved at Arg 133 to form a C-terminal 18KDa and an N-terminal 15KDa fragment due to the instability caused by a nearby Asp 137 in the hydrophobic ridge of the coiled-coil α-helix (Pato et al., 1981; Sumida et al., 2008). WT, Tm-D175N and Tm-E180G were also cleaved at R133, but Tm-D175N and Tm-E180G were cleaved 1.3X and 3.3X faster than WT, respectively. Cleavage of Tm bond to actin was slowed an order of magnitude with similar order of rate: E180G > D175N > WT.In the absence of actin, Tm-E180G, is cleaved at K233 (as well as R133), to produce intermediates of 27KDa and 6KDa. At longer times, the 18KDa fragment of all mutants gets cleaved at R167 to produce a 13.5KDa fragment. For Tm-E180G, the N-terminal 27KDa intermediate is similarly cut at R167 to produce the15KDa fragment and a new 12KDa fragment. ATPase studies of reconstituted thin filaments showed an elevated ATPase at low Ca2+ with: Tm-E180G > Tm-D175N > WT. Thus, whereas both Tm-D175N and Tm-E180G show greater instability near R133 than WT, for Tm-E180G there is an additional region of dynamic instability, near K233. The increased instability for both mutants would result in greater flexibility which appears to be involved in the increased Ca2+-sensitivity observed. Supported by NIH HL 91162.