A two-dimensional spin-diffusion NMR study on the local structure of a water-soluble model peptide for Nephila clavipes dragline silk (MaSp1) before and after spinning

INTRODUCTION The dragline silk of the golden orb web spider Nephila clavipes has received significant attention because of its remarkable mechanical properties, which include toughness and high tensile strength.1,2 This silk contains two structural proteins designated as major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2).3,4 The dominant MaSp1 can be described as AB block-copolymers consisting of numerous alternating poly-Ala (A) and Gly-rich (B) blocks. The dragline silk fiber is produced from an aqueous spinning solution (dope) containing B20% w/v spidroins at ambient pressure and temperature with substantial conformational change. Several factors have been thought to be important for controlling this conformational change, including mechanical strain, the concentration of fibroin, changes in pH, and the concentration of other ions.5,6 A pH change from 6.9±0.1 to 6.3±0.1 from the start of the silk gland duct to the spigot is crucial for gelling the fibroin, increasing its sensitivity to shear, and initiating the conformation transition.1,6,7 We previously performed solid-state NMR structural analysis of 13C selectively labeled versions of the water-soluble model peptide, (E)8GGLGGQGAG(A)6GGAGQGG-YGG, to gain insight about the local structure of the MaSpl protein before and after spinning.8 Polyglutamic acid (E)8 included at the amino terminus makes the peptide water soluble, leading to mimicking the predominance of amino acids with acidic side chains in this part of the sequence in spider fibroins and the induction of the conformational transition by lowering the pH.6,9 One-dimensional 13C-cross polarization magic angle-spinning NMR spectra of the lyophilized and acid-treated peptide samples revealed that the conformational change induced by acidification primarily occurred in the poly-Ala, and to a lesser extent in the GAGA and GGA sequences. Moreover, the amount of b-sheet was the largest in the center of the poly-Ala domain compared with its periphery. These observations suggest that the peptide can be used as a model for studying the effects of acidification on changes in the local conformation.8 However, further detailed structures, such as the torsion angles in the b-sheet fraction, are still unclear. Two-dimensional (2D) proton-driven spin-diffusion solid-state NMR under off-magic-angle spinning (OMAS) coupled with 13C isotope double labeling of specific residues has been used as a powerful method for determining the torsion angles of the backbone amino-acid residues for the silk model peptides.10–18 The combination with rotational-echo double-resonance measurements is a more quantitative approach, as previously reported,10,15,18 but it is not easy to quantitatively determine the structure when the structure is heterogeneous, such as a mixture of b-sheets and random coil conformations. In this case, it is better to first rigorously determine the fraction of the mixed conformations of individual residues. We should then try to determine the structure using spin-diffusion solidstate NMR by taking the fraction into account. In this paper, we will propose an analytical method for estimating the detailed local structures in the conformational ensemble system using 2D spin-diffusion OMAS NMR experiments combined with information about the fractions of different conformations at specific sites obtained from the 1D spectra.8 Furthermore, we will also discuss the detailed structures of the acid-treated model peptide, such as the torsion angles in the b-sheet component.