Holtzer M E, Kidd S G, Crimmins D L, Holtzer A
Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Protein Sci. 1992 Mar;1(3):335-41. doi: 10.1002/pro.5560010305.
Native tropomyosin from rabbit skeletal muscle (RSTm) consists mainly of alpha alpha and alpha beta coiled coils (alpha/beta approximately 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 = beta). In such cross-linked RSTm, 3-8% beta = 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 approximately 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 degrees C or by incubating at 45 degrees 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).
来自兔骨骼肌的天然原肌球蛋白（RSTm）主要由αα和αβ卷曲螺旋组成（α/β约为3 - 4/1）。在一些现有研究中，未发现ββ分子。在本研究中，对几种不同制剂的RSTm进行了二硫键交联，制剂和交联均在非变性条件下进行。检测交联产物中在C36和C190处均交联的ββ分子（β = β）的存在情况。在这种交联的RSTm中，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、C4反相高效液相色谱和自由溶液毛细管电泳实验检测到3 - 8%的β = β。当校正不完全双交联时，该百分比变为约4 - 10%的ββ，且与蛋白质浓度（0.1 - 10.0 mg/mL）无关，这表明观察到的ββ物种不是分子间交联产生的假象。通过加热至55℃、或在45℃孵育后淬冷至室温、或通过暴露于盐酸胍后通过透析进行逐步复性，变性后再复性时，ββ的比例增加，这表明重新缔合在一定程度上更有利于同二聚体形成而非随机缔合。该结果与当一条链上的巯基被羧酰胺甲基化时观察到的随机缔合不同（Holtzer，M.E.，Breiner，T.，& Holtzer，A.，1984，《生物聚合物》23，1811 - 1833），也与其他生物体原肌球蛋白报道的压倒性异二聚体偏好不同（Lehrer，S.S.，Qian，Y.，& Hvidt，S.，1989，《科学》246，926 - 928；Lehrer，S.S. & Qian，Y.，1990，《生物化学杂志》265，1134 - 1138）。
