Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 31;55(21):3048-59.
doi: 10.1021/acs.biochem.6b00429. Epub 2016 May 18.

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

Muzaddid Sarker  1 , Kathleen E Orrell  1 , Lingling Xu  1 , Marie-Laurence Tremblay  1 , Jessi J Bak  1 , Xiang-Qin Liu  1 , Jan K Rainey  1
Affiliations

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

Muzaddid Sarker et al. Biochemistry. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A) Superposed solution NMR structural ensemble of W1 (PDB entry 2MU3) showing side chains only for residues mutated to tryptophan. Putative structural ensembles for (B) R36W, (C) F90W, (D) F146W and (E) Y169W tryptophan mutants (20 lowest energy members of 100 member ensembles superposed onto the lowest energy structure; calculated using wild type W1 NMR restraints with removal of distance restraints involving the mutated side chain). 5-fluoro moieties are represented by blue spheres.
Figure 2
Figure 2
Far-UV CD spectra of fluorinated W1 tryptophan mutants (5F-Trp-labeled at R36W, F90W, F146W and Y169W) and wild type (WT) W1.
Figure 3
Figure 3
2D 1H-15N HSQC spectra of indicated W1 5F-Trp-labeled mutants overlaid on wild type (WT) W1. Note that some side chain 1H-15N cross-peaks are aliased differently in W1 WT vs. W1 mutants (WT W1 data were acquired previously using a different field strength and sweep witdth). Combined chemical shift perturbations as a function of position are mapped onto the lowest energy member of each calculated mutant protein structural ensemble, with the thickness of the cylinder representing the degree of 1H-15N HSQC peak displacement. Positions corresponding to cross-peaks that broadened beyond detection under experimental conditions are colored in magenta and ambiguous cross-peaks are colored in yellow.
Figure 4
Figure 4
Optical micrographs showing manually drawn fibers formed by wild type (WT) and mutant W2 proteins in indicated buffer (scale bars: 20 μm).
Figure 5
Figure 5
1D 19F-NMR spectra of W1 5F-Trp-labeled mutants and free 5-fluoroindole as observed in indicated H2O/D2O mixture (W1 mutants: 512 scans; 5-fluoroindole: 128 scans).
Figure 6
Figure 6
Titration of wild type W1 followed at indicated backbone amide site by heteronuclear NMR (A, D; combined chemical shift perturbation based on our previously reported data) and of indicated 5F-Trp-labeled mutants followed by 19F NMR, with absolute (B, E) and normalized (C, F) 19F chemical shift changes illustrated (spectra shown in Figure S8). W1-Avg. represents average from all assigned residues (± average deviation). Dashed lines are used to join titration points when signals could not be tracked due to severe line broadening.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Gosline JM, Guerette PA, Ortlepp CS, Savage KN. The mechanical design of spider silks: From fibroin sequence to mechanical function. J Exp Biol. 1999;202:3295–3303. - PubMed
    1. Vollrath F, Porter D. Spider silk as a model biomaterial. Appl Phys A Mater Sci. 2006;82:205–212.
    1. Tokareva O, Jacobsen M, Buehler M, Wong J, Kaplan DL. Structure-function-property-design interplay in biopolymers: Spider silk. Acta Biomater. 2014;10:1612–1626. - PMC - PubMed
    1. Pritchard EM, Valentin T, Panilaitis B, Omenetto F, Kaplan DL. Antibiotic-releasing silk biomaterials for infection prevention and treatment. Adv Funct Mater. 2013;23:854–861. - PMC - PubMed
    1. Wendt H, Hillmer A, Reimers K, Kuhbier JW, Schafer-Nolte F, Allmeling C, Kasper C, Vogt PM. Artificial skin--culturing of different skin cell lines for generating an artificial skin substitute on cross-weaved spider silk fibres. PLoS One. 2011;6:e21833. - PMC - PubMed

Publication types

LinkOut - more resources