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Forschungsarbeit

Structure, stability and assembly mechanism of spider silk proteins

Von Franz Hagn (07.09.2010)

Spider silk is one of the thoughest natural materials known so far. However, the mechanism of silk fiber formation is far from clear. How do spiders manage to store their highly concentrated silk protein material inside their body in a soluble form and still be able to transform it into a stable fiber within seconds? In our expectation, these proteins would immediately tend to aggregate and kill the animal. Silk proteins are reminiscent of amphiphilic block copolymers containing stretches of polyalanine and glycine-rich polar elements forming a repetitive core flanked by highly conserved non-repetitive amino-terminal and carboxy-terminal domains. The C-terminal domain was implicated in the control of solubility and fibre formation initiated by changes in ionic composition and mechanical stimuli known to align the repetitive sequence elements and promote b-sheet formation.

Here, the nuclear magnetic resonance (NMR) solution structure of the highly conserved C-terminal domain of a spider dragline silk protein is presented. The structure is a novel dimeric disulphide bridged five-helix bundle with interleaved segments and two salt bridges in each subunit (Fig. 1).

Fig. 1: NMR solution structure of the C-terminal domain of a dragline silk protein from the garden spider. The 20 lowest-energy structures are overlayed giving an r.m.s.d of 0.18 Å for all protein backbone atoms.[Bildunterschrift / Subline]: Fig. 1: NMR solution structure of the C-terminal domain of a dragline silk protein from the garden spider. The 20 lowest-energy structures are overlayed giving an r.m.s.d of 0.18 Å for all protein backbone atoms.

The structural state of this protein domain is controlled by chemical and mechanical stimuli, which appears to be essential for switching between the storage and the assembly form of silk proteins in a controlled manner. The protein is stabilized in the presence of high salt whereas it tends to aggregate when shear stress is applied (Fig. 2). Within the spider, silk proteins are stored in presence of high salt whereas during fibre assemly, shear stress is applied and the proteins tend to aggregate in a controlled manner. The C-terminal domain governs this process and prevents premature aggregation.

Fig. 2: The structure of the C-terminal domain is labile against shear stress and can be stabilized with salt.[Bildunterschrift / Subline]: Fig. 2: The structure of the C-terminal domain is labile against shear stress and can be stabilized with salt. a) The two salt bridges in each monomer were found to be the most labile part of the protein. b) Salt leads to an apparent stabilization of the protein. c) Shear stress is the crucial trigger of the aggregation process of the C-terminal domain.

Furthermore, this domain plays a key role in the alignment of secondary structural features formed by the repetitive sequence elements of spider silk proteins, which is known to be important for the mechanical properties of the fiber. Silk protein constructs lacking this domain form amorphous aggregates and do not show proper protein chain alignment that is a prerequisite for stable fiber formation (Fig. 3).

Fig. 3: Shear-induced fibre production using spider silk constructs conaining the C-terminal repetitive domain (upper panel) and without this domain (lower panel).[Bildunterschrift / Subline]: Fig. 3: Shear-induced fibre production using spider silk constructs conaining the C-terminal repetitive domain (upper panel) and without this domain (lower panel). Correct alignment of the repetitive sequence elements is can only be achieved when the C-terminal domain is present. Without the C-terminal domain, only protein aggregates are visible.

Therefore, this domain can be called a "molecular switch" that controlls the transition between silk protein storage and fiber assembly within the spider (Fig. 4).

 

Fig. 4: Model of the fibre assembly process.[Bildunterschrift / Subline]: Fig. 4: During silk protein storage, the silk proteins form higher oligomeric assemblies stabilized by salt. In this state the C-terminal domain (NR, non-repetitive) initiates this assembly process. Under shear stress these assembies undergo controlled aggregation and the C-terminal domain drives that process and leads to correct alignment of the repetitive protein chains.

Franz Hagn
Franz Hagn
* 1977, Bruckmühl

Stationen
  • since 2010
  • Postdoctoral fellow at Harvard Medical School, Boston, USA.
  • 2004-2009
  • PhD student at the Technische Universität München at the Institute for Organic Chemistry and Biochemistry.
  • Topic: "NMR Spectroscopy of Molecular Chaperones, the p53 Tumorsuppressor Network and Spider Silk Proteins".
  • 2003
  • Internship at Aventis Pharma Germany GmbH, Frankfurt am Main at the division Drug Innovation and Approval, Chemistry.
  • Topic: "Binding studies of compounds to Receptors and Enzymes by NMR-Spectroscopy".
  • 2002-2003
  • Diploma thesis at the University of Bayreuth. Topic: "Real time NMR-Spectroscopy for studying protein folding of Ribonuclease T1". Supervisor: PD Dr. Jochen Balbach.
  • 1998-2003
  • Study of Biochemistry at the University of Bayreuth, Germany.

Stipendien und Auslandsaufenthalte
  • since 2010
  • EMBO long-term fellowship for postdoctoral research
  • 2006-2009
  • PhD scholarship of the Elite Network of Bavaria.
  • 2002
  • Visiting student at the Department of Biochemistry and Biophysics at Stockholm University, Sweden.

Veröffentlichungen
  • *Hagn, F., Thamm, C., Scheibel, T. & Kessler, H. (2010): "pH dependent dimerisation and salt dependent stabilisation of the N-terminal domain of spider dragline silk - Implications for fibre formation". Angew. Chem. Intl. Ed., in revision.
  • *Hagn F., Eisoldt L., Hardy J.G., Vendrely C., Coles M., Scheibel T. & Kessler H. (2010): "A conserved spider silk domain acts as molecular switch that controls fibre assembly". Nature 465: 239-242.
  • *Hagn F., Klein C., Demmer O., Marchenko N., Vaseva A., Moll U.M. & Kessler H. (2010): "BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain". J. Biol. Chem. 285: 3439-3450.
  • *Retzlaff M., Hagn F., Mitschke L., Hessling M., Gugel F., Kessler H., Richter K. & Buchner J. (2010): "Asymmetric activation of the Hsp90 dimer by its co-chaperone Aha1". Mol. Cell 37: 344-354.
  • *Spichty M., Taly A., Hagn F., Kessler H., Barluenga S., Winssinger N. & Karplus M. (2009): "The HSP90 binding mode of a radicicol-like E-oxime determined by docking,binding free energy estimations, and NMR 15N chemical shifts. Biophys. Chem. 143: 111-23 .
  • *Feige M.J., Hagn F., Esser J., Kessler H. & Buchner J. (2007): "Influence of the internal disulfide bridge on the folding pathway of the CL antibody domain". J. Mol. Biol. 365: 1232-1244.
  • *Richter K., Moser S., Hagn F., Friedrich R., Hainzl O., Heller M., Schlee S., Kessler H, Reinstein J. & Buchner J. (2006): "Intrinsic inhibition of the Hsp90 ATPase activity". J. Biol. Chem. 281: 11301-11311.
  • *Scholz C., Eckert B., Hagn F., Harrschmidt P., Balbach J. & Schmid F.X. (2006): "SlyD Proteins from different species exhibit high prolyl isomerase and chaperone activities". Biochemistry 45: 20-33.
  • *Andersson, A., Almqvist, J., Hagn, F. & Mäler, L. (2004): "Diffusion and dynamics of penetratin in different membrane mimicking media". Biochimica et Biophysica Acta 1661: 18-25.