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, NationalUniversity of Singapore, 14 Science

Drive 4, Singapore 11754

Key words: egg case silk,NMR assignment, NMR spectroscopy,TuSp1

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Word count: 4701 characters
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 lowglycine content (Foelix, R. F. 1996). A novel silk cDNA clone (TuSp1) from the golden web spider Nephila antipodiana was recently isolatedby 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 C-terminus) shows more sequence divergence. Unlike other silk proteins, the repeat domainencoded 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, wereport nearly complete backbone and side-chainassignments 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.

Methods and experiments

The gene coding for the structural region ofTuSp1-RP1 (1-160) of TuSp1 was cloned into pET-M over-expression vector and over-expressed in the15N- or 15N/13C-labeled form in E. coli BL21 (DE3) growing in M9-minimal medium containing only 15N-labeled NH4Cl and 13C-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 pAminoBenzamidine-Agarose (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~1.0mM protein were prepared in 50 mM phosphate buffer, 2mM EDTA, and 50μM 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 (Delaglioet al., 1995) and analyzed with the softwareNMRView(Johnson and Blevins, 1994) on Linux workstations. Backbone resonance assignments were obtained with HNCACB, CBCA(CO)NH, 15N-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 NOESYand 2D 1H-13C HSQCexperiments. Aromatic side-chain spins were assigned from 13C-edited NOESY and 15N-edited NOESY.

Extent of assignments and data deposition

All the 1H and 15N backbone resonances were assignedexcept for the first2 amino acids from the expression vector, for which signals could not bedetected in the1H-15N HSQC spectrum.More than 94% of the side-chain 1H and 13Cresonances (including both aliphatic and aromatic spins) were assigned. Side-chain NH2 from Gln and Asn are partially assigned. Figure 1shows the 1H-15N HSQC spectrum of the uniformly 15NenrichedTuSp1-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 ( under accession number BMRB-6864.

Acknowledgements

This research has been supported by theNational University of Singapore to D.W.Y.

References

Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) J. Biomol. NMR, 6, 277–293.

Foelix, R. F., The Biology of spiders. New York: OxfordUniversity 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)Biochemistry44, 10020-10027.

Johnson, B.A. and Blevins, R.A. (1994) J. Biomol. NMR, 4, 603–614.

Tian, M. and Lewis, R. V., (2005) Biochemistry44, 8006-8012.

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, 100mM NaCl, 2mM EDTA, pH 6.5,50μM sodium azide. Gln/Asn side-chain cross peaks are denoted with horizontal lines. Unassigned sidechain resonances are indicated by asterisks (*).

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