Segmented nanofibers of spider dragline silk: Atomic force microscopy and single-molecule force spectroscopy

doi:10.1073/pnas.082526499

Segmented nanofibers of spider dragline silk: Atomic force microscopy and single-molecule force spectroscopy

^*Department of Physics, University of California, Santa Barbara, CA 93106; and ^†U.S. Army Natick R&D Center, Natick, MA 01760

Edited by Ignacio Tinoco, Jr., University of California, Berkeley, CA, and approved February 5, 2002 (received for review October 3, 2001)

Abstract

Despite its remarkable materials properties, the structure of spider dragline silk has remained unsolved. Results from two probe microscopy techniques provide new insights into the structure of spider dragline silk. A soluble synthetic protein from dragline silk spontaneously forms nanofibers, as observed by atomic force microscopy. These nanofibers have a segmented substructure. The segment length and amino acid sequence are consistent with a slab-like shape for individual silk protein molecules. The height and width of nanofiber segments suggest a stacking pattern of slab-like molecules in each nanofiber segment. This stacking pattern produces nano-crystals in an amorphous matrix, as observed previously by NMR and x-ray diffraction of spider dragline silk. The possible importance of nanofiber formation to native silk production is discussed. Force spectra for single molecules of the silk protein demonstrate that this protein unfolds through a number of rupture events, indicating a modular substructure within single silk protein molecules. A minimal unfolding module size is estimated to be around 14 nm, which corresponds to the extended length of a single repeated module, 38 amino acids long. The structure of this spider silk protein is distinctly different from the structures of other proteins that have been analyzed by single-molecule force spectroscopy, and the force spectra show correspondingly novel features.

Footnotes

↵ ‡ To whom reprint requests should be addressed. E-mail: hhansma{at}physics.ucsb.edu.
This paper results from the Arthur M. Sackler Colloquium of the National Academy of Sciences, “Nanoscience: Underlying Physical Concepts and Phenomena” held May 18–20, 2001, at the National Academy of Sciences in Washington, DC.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

AFM,

atomic force microscope/microscopy;

(GGX)n,

repeating aa sequences glycine-glycine-X, where X is usually leucine, tyrosine, or glutamine, represented as a loose or tight spiral in Figs. 3A and 5A and B;

H-bond,

hydrogen bond;

poly(A/GA),

aa sequences of poly(alanine)/poly(glycylalanine), represented as a zig-zag in Figs. 3A and 5 A and B;

poly(A/GA+GGX),

a poly(A/GA) sequence of SPI plus the (GGX)n sequence of SPI that follows it: GAGAAAAAAAAAAGGAGQGGYGGLGSQGTSGRGGLGGQ;

pS(4+1),

modular recombinant spider silk protein composed of 16 SPI and 4 SPII modules arranged as follows: (SPI)4-SPII-(SPI)4-SPII-(SPI)4-SPII-(SPI)4-SPII (Fig. 1);

SPI,

38-aa-long synthetic spider silk sequence based on sequence from Nephila clavipes: SGRGGLGGQGAGAAAAAAAAAAGGAGQGGYGGLGSQGT;

SPII,

12-aa-long synthetic spider silk sequence based on sequence from N. clavipes: SGPGGYGPGQQT;

WLC,

worm-like chain (polymer model)