Supplementary Material
Resonance Assignments of a Repeated Domain of the Egg Case Silk from
Nephila Antipodiana
Zhi Lin, Weidong Huang & Daiwen Yang∗
Department of Biological Sciences, National University of Singapore, 14 Science
Drive 4, Singapore 11754
Key words: egg case silk, NMR assignment, NMR spectroscopy, TuSp1
*To whom correspondence should be addressed, E-mail: dbsydw@nus.edu.sg
Tel: +65-68741014
Fax: +65-67792486
Word count: 4701 characters
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Biological context
Spider silks are renowned for their excellent mechanical properties. Although
several spider fibroin genes, mainly from dragline and capture silks, have been
identified (Gatesy, J et al., 2001), the exact number and composition of the spider
fibroin gene family remain unclear. Among seven different silks produced by
different abdominal glands for various functions, tubuliform silk (eggcase silk) is
unique due to its high serine and low glycine content (Foelix, R. F. 1996). A novel
silk cDNA clone (TuSp1) from the golden web spider Nephila antipodiana was
recently isolated by us (Huang et al. submitted). Both In situ hybridization and
immunoblot analyses have showed that it is specifically expressed in the tubuliform
gland. The translated sequence of TuSp1 accounts for the amino acid composition of
tubuliform silk fibers very well, indicating that TuSp1 is the major component of
tubuliform gland. On the basis of amino acid sequence alignment analysis with other
species (Tian et al., 2005; Hu et al., 2005; Garb et al., 2005), the putative repeat
sequence (designated as TuSp1-RP1) of TuSp1 in N. antipodiana was found to
consist of 170 residues. Identical repeats may extend for several more units from the
N-terminus. The last 56 amino acid residues in the last repeat (nearest to the Cterminus) shows more sequence divergence. Unlike other silk proteins, the repeat
domain encoded by the novel cDNA in water solution exhibits the characteristic of an
-helical structure. The unique structure implies the distinct property of the egg case
silk. Its sequence information also facilitates elucidation of the evolutionary history of
the araneoid fibroin genes.
Here, we report nearly complete backbone and side-chain assignments of the
repeated domain from TuSp1 as a first step towards a better understanding of the
relationship between the amino acid sequence and silk property.
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Methods and experiments
The gene coding for the structural region of TuSp1-RP1 (1-160) of TuSp1 was
cloned into pET-M over-expression vector and over-expressed in the 15N- or 15N/13Clabeled form in E. coli BL21 (DE3) growing in M9-minimal medium containing only
15
N-labeled NH4Cl and
13
C-labeled glucose as the sole nitrogen and carbon source.
The protein was purified by immobilized metal affinity chromatography on Ni-NTA.
The N-terminal His-tag was cleaved by thrombin. Ni-NTA and pAminoBenzamidineAgarose (Sigma) were used to remove the His-tag and thrombin. Gel filtration was
used to purify the protein. The resulting protein contains two additional residues
(Gly-Ser) from the vector’s cleavage site. NMR samples containing ~1.0 mM protein
were prepared in 50 mM phosphate buffer, 2 mM EDTA, and 50 μM sodium azide at
pH 6.5. NMR experiments were performed at 290 K on a Bruker Avane 800MHz
spectrometer.
All NMR data were processed with NMRPipe software (Delaglio et al., 1995)
and analyzed with the software NMRView (Johnson and Blevins, 1994) on Linux
workstations. Backbone resonance assignments were obtained with HNCACB,
CBCA(CO)NH,
15
N-edited NOESY and 1H-15N HSQC experiments. Degeneracy in
(13C,13C) shifts was resolved by sequential 1HN-1HN NOEs. Side-chain assignments
were based on H(CCCO)NH, CC(CO)NH, 13C-edited NOESY and 2D 1H-13C HSQC
experiments. Aromatic side-chain spins were assigned from
15
13
C-edited NOESY and
N-edited NOESY.
Extent of assignments and data deposition
All the 1H and
15
N backbone resonances were assigned except for the first 2
amino acids from the expression vector, for which signals could not be detected in the
3
1
H-15N HSQC spectrum. More than 94% of the side-chain 1H and
13
C resonances
(including both aliphatic and aromatic spins) were assigned. Side-chain NH2 from
Gln and Asn are partially assigned. Figure 1 shows the 1H-15N HSQC spectrum of the
uniformly
15
N enriched TuSp1-RP1. Analysis of Cα, Cβ, NH and Hα chemical shifts
has established that the protein consists of 6 -helices and the first 15-residues and
last 25-residues exist in a random coil form. The assignments have been deposited in
the BioMagRes-Bank (http://www.bmrb.wisc.edu) under accession number BMRB6864.
Acknowledgements
This research has been supported by the National University of Singapore to D.W.Y.
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References
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Biomol. NMR, 6, 277–293.
Foelix, R. F., The Biology of spiders. New York: Oxford University Press, 1996.
Garb, J.E. and Hayashi, C.Y., (2005) Proc Natl Acad Sci, 102(32):11379-84.
Gatesy, J., Hayashi, C., Motriuk, D., Woods, J. and Lewis, R. (2001). Science 291,
2603-2605.
Hu, X., Lawrence, B., Kohler, K., Falick, A.M., Moore, A.M.F., McMullen, E., Jones,
P.R., and Vierra, C., (2005)Biochemistry 44, 10020-10027.
Johnson, B.A. and Blevins, R.A. (1994) J. Biomol. NMR, 4, 603–614.
Tian, M. and Lewis, R. V., (2005) Biochemistry 44, 8006-8012.
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Figure legend
Figure 1. . 1H-15N HSQC spectrum of TuSp1-RP1 acquired at 800 MHz and 290 K on
a sample of ~1 mM protein, 50 mM phosphate, 100 mM NaCl, 2 mM EDTA, pH 6.5,
50 μM sodium azide. Gln/Asn side-chain cross peaks are denoted with horizontal
lines. Unassigned sidechain resonances are indicated by asterisks (*).
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