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

Abstract

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, (19)F 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 (1)H-(15)N 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 (19)F chemical shift, line width, longitudinal relaxation time constant (T1), and solvent isotope shift. Perturbation to (19)F 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). (19)F NMR spectroscopy allowed unambiguous localized tracking throughout titration with each perturbant, demonstrating distinct behavior for each perturbant not previously revealed by heteronuclear NMR experiments.

Recombinant Silk Fiber Properties Correlate to Prefibrillar Self‐Assembly

“…As a whole, the α‐helical structuring of aciniform spidroin in solution appears relatively insensitive to environment. The fact that W 3 protein solubilized in all of these conditions formed fibers exhibiting a structural conversion from α‐helix to β‐sheet suggests that α‐helical structuring provides a stable aciniform spidroin form for storage in solution and a favorable starting point for structural transition under fiber‐forming conditions.…”

Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

“…Chaotropic denaturation or treatment with the detergent dodecylphopshocholine lead to helix 5 destabilization and a concomitant structural rearrangement in the globular core of the W unit [19,22]. Notably, therefore, the spectral density in helix 5 deviates from the remainder of the globular domain.…”

“…During fibre formation, AcSp1 undergoes a partial conversion from α-helical to β-sheet structuring [21], putatively seeded at helix 5 in the W unit [19,22]. This transition is recapitulated in recombinant W 2 between the soluble and fibrous forms [19].…”

Fluorotryptophan Incorporation Modulates the Structure and Stability of Transthyretin in a Site-Specific Manner

“…$$1/T_1=\frac{K^2}{20}\rho \left(\frac{4}{3}\pi r_{min}^{-3}\right)\left(J\left({\omega }_{\mathrm{normalH}}-{\omega }_{\mathrm{normalF}}\right)+3J\left({\omega }_{\mathrm{F}}\right)+6J\left({\omega }_{\mathrm{normalH}}+{\omega }_{\mathrm{normalF}}\right)\right)+\frac{6}{40}{\omega }_{\mathrm{normalF}}^2{\delta }_z^2\left(1+\frac{{\eta }^2}{3}\right)J\left({\omega }_{\mathrm{F}}\right)$$ $$1/T_2=\frac{K^2}{40}\rho \left(\frac{4}{3}\pi r_{min}^{-3}\right)\left(J\left({\omega }_{\mathrm{normalH}}-{\omega }_{\mathrm{normalF}}\right)+3J\left({\omega }_{\mathrm{F}}\right)+6J\left({\omega }_{\mathrm{normalH}}+{\omega }_{\mathrm{normalF}}\right)+4J\left(0\right)+6J\left({\omega }_{\mathrm{H}}\right)\right)+\frac{1}{40}{\omega }_{\mathrm{normalF}}^2{\delta }_z^2\left(1+\frac{{\eta }^2}{3}\right)\left(3J\left({\omega }_{\mathrm{F}}\right)+4J\left(0\right)\right)$$ where ρ is the density for proton-containing spheres in proteins as 5.73 ×10 28 m −3 from Sarker et al, 32 r min is the minimal distance from the nearest proton (2.6 Å) used for the spherical integral taken from Luck et al 31 J (ω) is the spectral density functions described by Abragam 33 sampled at the angular frequencies of 19 F or 1 H nuclei ( ω H or ω F at 14.1 T in our work) or linear combinations thereof, δ z is the chemical anisotropy for 19 F-Trp, η is the corresponding asymmetry and K = μ 0 γ H γ F ħ/4 π , where μ 0 is the vacuum permeability constant, γ x is the magnetogyric ratio for nucleus x and ħ is the reduced Planck constant. For 5FW, δ z = −62.3 ppm and η = 0.24.…”