Effects of an interchain disulfide bond on tropomyosin structure: intrinsic fluorescence and circular dichroism studies |
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Authors: | S S Lehrer |
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Affiliation: | 1. Department of Muscle Research Boston Biomedical Research Institute Boston, Mass. 02114, U.S.A.;2. Department of Neurology Harvard Medical School Boston, Mass., U.S.A. |
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Abstract: | An interchain disulfide crosslink was introduced into rabbit skeletal tropomyosin (TM) at Cys190 by two different methods under non-denaturing conditions. The effects of the crosslink on the structure of tropomyosin were investigated by fluorescence and circular dichroism methods as a function of temperature and guanidine · hydrochloride concentration. Four different preparations were studied: Nbs2-TM, red-TM crosslinked with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate); O2-TM, TM whose SH groups were air-oxidized; red-TM, TM reduced with dithiothreitol; IA-TM, red-TM whose SH groups were blocked with iodoacetamide. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies indicated that SS crosslinks were quantitatively introduced between the subunits of TM for Nbs2-TM and O2-TM. In the completely folded state (below 25 °C or in the absence of denaturant) and in the unfolded state (above 65 °C or greater than 4 m-guanidine · hydrochloride) all of the samples had the same Tyr fluorescence quantum yield, accessibility to acrylamide fluorescence quenching, fluorescence polarization and mean residue rotation at 222 nm. Thermal and denaturant-induced unfolding profiles at pH 7.5 were obtained for each sample with measurements of these parameters. The main transition at about 45 °C or 2 m-guanidine · hydrochloride was shifted about +7 deg. C and 0.8 m in guanidine · hydrochloride, respectively, for the crosslinked samples as compared to the uncrosslinked samples. In addition, a destabilizing pretransition was observed in the 30 to 45 °C region or the 0 to 2 m-guanidine · hydrochloride region only for the crosslinked samples when polarization or ellipticity was measured. Studies of the ability of Nbs2 to crosslink red-TM as a function of guanidine · hydrochloride concentration indicated that the chains separate at Cys190 between 0 and 2 m-guanidine · hydrochloride before they dissociate. Thus, the effect of the SS crosslink at Cys190 on the conformation of TM at physiological temperatures appears to be related to the inherent instability of the molecule in this region of the sequence. |
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Keywords: | Addresssed for correspondence |
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