Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

Abstract

Spider aciniform (wrapping) silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units). In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR) spectroscopy-based 15N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps–ns timescale in the context of the single W unit (W1) and the two unit concatemer (W2). Unambiguous mapping of backbone dynamics throughout W2 was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W1 and W2 reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre.

Structural and Mechanical Roles for the C-Terminal Nonrepetitive Domain Become Apparent in Recombinant Spider Aciniform Silk

“…Prominent negative bands at 208 and 220 nm, along with strongly positive ellipticity at 195 nm, indicate that all proteins contain significant α-helical content in solution. This is consistent with our solution-state NMR structures of W 1 and W 2 , showing each W unit to contain a globular domains of 5 α-helices connected to its neighbor by an intrinsically disordered linker. ,, All CTD structures reported to date also, coincidentally, contain 5 helices followed by a disordered segment. ,,, CD spectra representative of a convolution of helical and disordered structures is therefore fully expected, but the exceptional degree of similarity of mean residue ellipticity for all four protein constructs is striking and would not be predicted a priori. As a whole, it is clear that swapping of a W unit for a CTD does not perturb the structuring or independence of the repetitive units in solution and also that CTDs and W unit have a similar overall structural composition.…”

“…The main component of aciniform silk is AcSp1 (aciniform spider silk protein 1, spidroin 1), which is produced and stored in the aciniform gland as a highly soluble protein and can be readily spun into solid fibers. From solution to a solid-state fiber, AcSp1 undergoes a structural transition from globular helical domains connected by intrinsically disordered linkers , to a similar proportion of disorder alongside a mixture of oriented β-sheet and α-helical domains . α-Helix to β-sheet conversion is believed to provide strength to silk fibers. , …”

NMR Structure and Biophysical Characterization of Thermophilic Single-Stranded DNA Binding Protein from Sulfolobus Solfataricus

“…From Figure 6 c, we can observe higher R 2 /R 1 values at 25 °C than 50 °C. The average tumbling time (τ c ) values calculated from R 2 /R 1 were 12.657 ns and 5.012 ns at 25 °C and 50 °C, respectively [ 40 , 41 ], indicating that the protein tumbles twice as fast at high temperature as it does at room temperature.…”

“…From the R 1 and R 2 data, it was confirmed that the residues that experience fast dynamics were more prominent at 50 • C, and the overall motion of the protein was faster at a higher temperature. From Figure 6c, we can observe higher R 2 /R 1 values at 25 • C than 50 • C. The average tumbling time (τ c ) values calculated from R 2 /R 1 were 12.657 ns and 5.012 ns at 25 • C and 50 • C, respectively [40,41], indicating that the protein tumbles twice as fast at high temperature as it does at room temperature.…”

“…From Figure 6c, we can observe higher R2/R1 values at 25 °C than 50 °C . The average tumbling time (τc) values calculated from R2/R1 were 12.657 ns and 5.012 ns at 25 °C and 50 °C , respectively [40], [41], indicating that the protein tumbles twice as fast at high temperature as it does at room temperature. From the hetNOE data (Figure 6d), residues T32, N34, G35, and V36 located in L12; residues S97 and D99 in the loop between β5 and H3; N110 at the C-terminus had hetNOE values lower than 0.6 at 25 °C.…”