Intradomain Distances in the Regulatory Domain of the Myosin Head in Prepower and Postpower Stroke States: Fluorescence Energy Transfer

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• 作者：Thomas Palm ; Ken Sale ; Louise Brown ; Huichun Li ; Brett Hambly ; and Peter G. Fajer
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：October 5, 1999
• 年：1999
• 卷：38
• 期：40
• 页码：13026 - 13034
• 全文大小：329K
• 年卷期：v.38,no.40(October 5, 1999)
• ISSN：1520-4995

文摘

The relative movement of the catalytic and regulatory domains of the myosin head (S1) islikely to be the force generating conformational change in the energy transduction of muscle [Rayment,I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993)Science 261, 58-65]. To test this model we have measured, using frequency-modulated FRET, threedistances between the catalytic domain and regulatory domains and within the regulatory domain of myosin.The donor/acceptor pairs included MHC cys707 and ELC cys177; ELC cys177 and RLC cys154; andELC cys177 and gizzard RLC cys108. The IAEDANS (donor) or acceptor (DABMI or IAF) labeled lightchains (ELC and RLC) were exchanged into monomeric myosin and the distances were measured in theputative prepower stroke states (in the presence of MgATP or ADP/AlF₄^-) and the postpower strokestates (ADP and the absence of nucleotides). For each of the three distances, the donor/acceptor pairswere reversed to minimize uncertainty in the distance measured, arising from probe orientational factors.The distances obtained from FRET were in close agreement with the distances in the crystal structure.Importantly, none of the measured distances varied by more than 2 Å, putting a strong constraint on theextent of conformational changes within S1. The maximum axial movement of the distal part of myosinhead was modeled using FRET distance changes within the myosin head reported here and previously.These models revealed an upper bound of 85 Å for a swing of the regulatory domain with respect to thecatalytic domain during the power stroke. Additionally, an upper bound of 22 Å could be contributed tothe power stroke by a reorientation of RLC with respect to the ELC during the power stroke.