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Protein Science, Vol 1, Issue 3 335-341, Copyright © 1992 by Cold Spring Harbor Laboratory Press


ARTICLE

{beta}{beta} homodimers exist in native rabbit skeletal muscle tropomyosin and increase after denaturation-renaturation

M. E. HOLTZER, S. G. KIDD, D. L. CRIMMINS and A. HOLTZER
Department of Chemistry, Box 1134, Washington University, One Brookings Dr., St. Louis, Missouri 63130

Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of {alpha}{alpha} and {alpha}{beta} coiled coils ({alpha}/{beta} {complex} 3-4/1). In some extant studies, no {beta}{beta} molecules have been found. In this study, RSTm from several different preparations was disulfide cross-linked, both preparation and cross-linking being done under nondenaturing conditions. The cross-linked product was assayed for the presence of {beta}{beta} molecules cross-linked at both C36 and C190 ({beta}{complex}{beta}). In such cross-linked RSTm, 3-8% {beta}{complex}{beta} is detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, C4 reversed-phase high-performance liquid chromatography, and a free-solution capillary electrophoresis experiment. This percentage becomes ~4-10% {beta}{beta} when corrected for incomplete double cross-linking and is independent of protein concentration (0.1-10.0 mg/mL), indicating that the observed {beta}{beta} species are not artifacts due to intermolecular cross-linking. Upon denaturation and subsequent renaturation either by heating to 55{deg}C or by incubating at 45{deg}C followed by quenching to room temperature, or by guanidine hydrochloride exposure followed by phased renaturation by dialysis, the fraction of {beta}{beta} increases, indicating that the reassociation favors homodimer formation somewhat over random association. This result differs from the random association observed when the sulfhydryl on one of the chains is carboxyamidomethylated (Holtzer, M.E., Breiner, T., & Holtzer, A., 1984, Biopolymers 23, 1811-1833), and from the overwhelming heterodimer preferences reported for tropomyosins from other organisms (Lehrer, S.S., Qian, Y., & Hvidt, S., 1989, Science 246, 926-928; Lehrer, S.S. & Qian, Y., 1990, J. Biol. Chem. 265, 1134-1138).
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