Tracking Transitions in Spider Wrapping Silk Conformation and Dynamics by 19F Nuclear Magnetic Resonance Spectroscopy

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• 作者：Muzaddid Sarker ; Kathleen E. Orrell ; Lingling Xu ; Marie-Laurence Tremblay ; Jessi J. Bak ; Xiang-Qin Liu ; Jan K. Rainey
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：May 31, 2016
• 年：2016
• 卷：55
• 期：21
• 页码：3048-3059
• 全文大小：674K
• 年卷期：0
• ISSN：1520-4995

文摘

Aciniform silk protein (AcSp1) is the primary component of wrapping silk, the toughest of the spider silks because of a combination of high tensile strength and extensibility. Argiope trifasciata AcSp1 contains a core repetitive domain with at least 14 homogeneous 200-amino acid units (“W” units). Upon fibrillogenesis, AcSp1 converts from an α-helix-rich soluble state to a mixed α-helical/β-sheet conformation. Solution-state nuclear magnetic resonance (NMR) spectroscopy allowed demonstration of variable local stability within the W unit, but comprehensive characterization was confounded by spectral overlap, which was exacerbated by decreased chemical shift dispersion upon denaturation. Here, ¹⁹F NMR spectroscopy, in the context of a single W unit (W₁), is applied to track changes in structure and dynamics. Four strategic positions in the W unit were mutated to tryptophan and biosynthetically labeled with 5-fluorotryptophan (5F-Trp). Simulated annealing-based structure calculations implied that these substitutions should be tolerated, while circular dichroism (CD) spectroscopy and ¹H–¹⁵N chemical shift displacements indicated minimal structural perturbation in W₁ mutants. Fiber formation by W₂ concatemers containing 5F-Trp substitutions in both W units demonstrated retention of functionality, a somewhat surprising finding in light of sequence conservation between species. Each 5F-Trp-labeled W₁ exhibited a unique ¹⁹F chemical shift, line width, longitudinal relaxation time constant (T₁), and solvent isotope shift. Perturbation to ¹⁹F chemical shift and nuclear spin relaxation parameters reflected changes in the conformation and dynamics at each 5F-Trp site upon addition of urea and dodecylphosphocholine (DPC). ¹⁹F NMR spectroscopy allowed unambiguous localized tracking throughout titration with each perturbant, demonstrating distinct behavior for each perturbant not previously revealed by heteronuclear NMR experiments.