Institute of Experimental Biology, Kazimierz Wielki University , Bydgoszcz , Poland.
PeerJ. 2013 Oct 15;1:e181. doi: 10.7717/peerj.181. eCollection 2013.
Tropomyosins are actin-binding regulatory proteins which overlap end-to-end along the filament. High resolution structures of the overlap regions were determined for muscle and non-muscle tropomyosins in the absence of actin. Conformations of the junction regions bound to actin are unknown. In this work, orientation of the overlap on actin alone and on actin-myosin complex was evaluated by measuring FRET distances between a donor (AEDANS) attached to tropomyosin and an acceptor (DABMI) bound to actin's Cys374. Donor was attached to the Cys residue introduced by site-directed mutagenesis near the C-terminal half of the overlap. The recombinant alpha-tropomyosin isoforms used in this study - skeletal muscle skTM, non-muscle TM2 and TM5a, and chimeric TM1b9a had various amino acid sequences of the N- and C-termini involved in the end-to-end overlap. The donor-acceptor distances calculated for each isoform varied between 36.4 Å and 48.1 Å. Rigor binding of myosin S1 increased the apparent FRET distances of skTM and TM2, but decreased the distances separating TM5a and TM1b9a from actin. The results show that isoform-specific sequences of the end-to-end overlaps determine orientations and dynamics of tropomyosin isoforms on actin. This can be important for specificity of tropomyosin in the regulation of actin filament diverse functions.
原肌球蛋白是肌动蛋白结合调节蛋白，沿纤维从头到尾重叠。在没有肌动蛋白的情况下，确定了肌肉和非肌肉原肌球蛋白的重叠区域的高分辨率结构。与肌动蛋白结合的连接区域的构象未知。在这项工作中，通过测量附着在原肌球蛋白上的供体（AEDANS）和结合在肌动蛋白的 Cys374 上的受体（DABMI）之间的 FRET 距离，评估了在单独肌动蛋白和肌球蛋白复合物上重叠的方向。供体附着在通过定点突变引入的 Cys 残基上，该残基位于重叠的 C 端一半附近。本研究中使用的重组α-原肌球蛋白同工型 - 骨骼肌 skTM、非肌肉 TM2 和 TM5a 以及嵌合 TM1b9a - 具有涉及头尾重叠的 N-和 C-末端的各种氨基酸序列。每种同工型计算出的供体-受体距离在 36.4 Å 和 48.1 Å 之间变化。肌球蛋白 S1 的严格结合增加了 skTM 和 TM2 的表观 FRET 距离，但降低了 TM5a 和 TM1b9a 与肌动蛋白之间的距离。结果表明，头尾重叠的同工型特异性序列决定了原肌球蛋白同工型在肌动蛋白上的取向和动力学。这对于原肌球蛋白在调节肌动蛋白丝多样化功能的特异性可能很重要。
