Changes in the Local Structure of Nephila clavipes Dragline Silk

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Changes in the Local Structure of Nephila clavipes Dragline Silk Model Peptides upon Trifluoroacetic Acid, Low pH, Freeze Drying and Hydration Treatments studied by C Solid State NMR 13

Tetsuo Asakura, Hironori Matsuda, Naomi Kataoka, and Akiko Imai Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01267 • Publication Date (Web): 03 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018

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Biomacromolecules

Changes in the Local Structure of Nephila clavipes Dragline Silk Model Peptides upon Trifluoroacetic Acid, Low pH, Freeze Drying and Hydration Treatments studied by 13C Solid State NMR

Tetsuo Asakura,* † Hironori Matsuda, † Naomi Kataoka † and Akiko Imai †

†

Department of Biotechnology, Tokyo University of Agriculture and Technology,

Koganei, Tokyo 184-8588 JAPAN

*Correspondence to: Tetsuo Asakura Tel & FAX: +84-42-383-7733 Email: [email protected]

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Abstract The conformational analysis of spider dragline silks is difficult because of the amorphous character of the silks. In this paper, the fractions of several conformations were determined for three 47-mer peptides, (Glu)4(Ala)6GlyGly12Ala13Gly14GlnGlyGlyTyrGlyGlyLeuGlySerGlnGly25Ala26Gly27ArgGlyGlyLeuGlyGlyGlnGly35Ala36Gly37(Ala)6(Glu)4 with three underlined 13C labeled blocks using 13C CP/MAS NMR method. The conformations of the 13C labeled sites change significantly depending on the location of the labelled blocks when treated with trifluoroacetic acid, low pH and freeze dried. The conformations of Ala36 and Gly37 residues are strongly influenced by specific conformation of (Ala)6 sequence at the C-terminal side, but those of other residues, Ala13 and Gly14, and Ala 26 and Gly27, are basically not influenced by the conformations of (Ala)6. Through hydration of the -sheet peptide, sharp peaks with random coil could be observed depending on the position of the residue, and this result could be interpreted via the change in the Ramachandran map obtained from molecular dynamics simulation.

Key words: Spider dragline silk / Sequential model peptide / 13C Solid-state NMR / Conformational analysis / Hydration


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Introduction Spider dragline silks continue to attract of many researchers because of their superior mechanical properties and many potential applications.1-5 One of the most studied spider silks is golden weaver Nephila clavipes (N. clavipes) dragline silk which contains two kinds of proteins, designated major ampullate spidroin 1 (MaSp1) and spidroin 2 (MaSp2).6-8 The repetitive domain of dominant MaSpl protein is mainly composed of polyalanine (poly-Ala) and Gly-rich motifs. The poly-Ala motif is known to form antiparallel -sheets (AP-) nanostructures that are responsible for the silk’s strength, and Gly-rich motif is responsible for the extensibility of the spider silk fiber. In addition, supercontraction is known as another unique character which occurs in the hydration process of the spider dragline silk fiber, i.e., the fiber causes to contract up to 50% in length as a result of interaction with water molecules.9-13 The informations about the structure and dynamics of several silks have been obtained using solid state NMR,14-30 IR,31-33 Raman 34-42 and X-ray diffraction methods43-47 experimentally. However, it is by no means easy to determine the atomic-level structure because the spider silk proteins have complex and amorphous nature. The most detailed picture about the atomic-level structure and dynamics of the silks has come from NMR spectroscopy, using both solid and solution state measurements. In our previous papers,26,48,49 for the first time we could determine the packing structures of AP- poly-Ala motif in spider silks in both dry and hydrated states. The use of a series of alanine oligopeptides as the model for poly-Ala motif was very useful because the Ala C peak in



the 13C cross-polarization/magic angle spinning (CP/MAS) NMR spectrum of AP- (Ala)6 was typical of the rectangular packing structure, and that of AP- (Ala)7 was quite different and typical of the staggered packing structure.48 The Ala C peaks in the 13C CP/MAS NMR spectra of spider dragline silks were reproduced well by assuming a mixture of rectangular and staggered packing structures, and the fractions of them were determined using mainly 13C CP/MAS NMR methods.49 Here the atomic level structures of both rectangular and staggered packing structures were also delineated.48,49 In contrast, it is more difficult to determine the conformation of the amorphous Gly-rich motif because there are irregular amino acid sequences which cause a lot of variations in the local conformations. The 13C labeling of spider silk proteins by administering 13C labeled amino acids in aqueous solution in the mouth of the spiders directly or administering 13C labeled cricket has been reported and yielded a lot of structural information.21-24 However, these 13C labeling methods are limited because 13C selective labeling of the individual residues in Gly-rich motif is difficult. Despite this difficulty, it is very helpful to use solid state NMR and the selectively stable-isotope labeled model peptides are useful for conformational analysis of the Gly-rich motif.48-52 Indeed, we have synthesized the model peptide which was carefully designed from the primary structure of N. clavipes dragline silk with the goal of determining the fraction of each residue conformation in the Gly-rich motif.52 Thus, the 13C-selectively

labeled 47-mer peptides were designed with the primary structure,

(E)4(A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6(E)4 (Glu (E), Ala (A), Gly (G), Gln (Q), Tyr (Y), Leu (L), Ser (S) and Arg (R)). It was synthesized where three 13C labeled amino acid blocks, [2-13C]Gly12[3-13C]Ala13[1-13C]Gly14 , [2-13C]Gly25 [3-13C]Ala26[1-


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13C]Gly27

and [2-13C]Gly35[3-13C]Ala36[1- 13C]Gly37 were introduced as shown in

Supporting Information (Table 1S), together with seven [1-13C]Gly and [15N]Gly-double labeled 47-mer peptides with different 13C and 15N labeled positions. The 13C CP/MAS NMR spectra were observed for determination of the fractions of the several conformations adopted by each 13C labeled residue. Useful information included the conformation-dependent 13C solid state NMR chemical shifts and the peak deconvolution of 13C labeled sites in these peptides.53-56 Here two (Glu)4 blocks at both termini were attached for making them soluble in water. The aqueous solutions of the peptides could be precipitated by lowering the pH to 4. By this low pH treatment, the conformation of the poly-Ala sequence became AP- sheet structure, which was a model of the silk fiber after spinning.51,52,57,58 Moreover, the results obtained experimentally in our study were able to explain successfully through molecular dynamics (MD) calculation.52 From these studies, we could disprove the presence of 31 helix in the Gly-rich motif of spider silk fibers although the presence of such a structure has been frequently reported from 13C solid state NMR experiments.22,59-62 Thus, we could determine the detailed local conformation of the Gly-rich motif in spider silk fiber and verify previously reported discussions about the conformation of the Gly-rich motif using 13C-selectively labeled 47-mer peptides with AP- sheet poly-Ala structure in the previous paper.52 In this paper, we used three 13C-selectively labeled 47-mer peptides with the primary structure, (E)4(A)6GGAGQGGYGGLGSQGAGRGGLGGQGAG(A)6(E)4 again to clarify the structural and dynamical changes in the spider dragline silks treated with different conditions such as trifluoroacetic acid (TFA), low pH, or freeze drying treatments together 5 ACS Paragon Plus Environment


with the effect of hydration on the conformation and dynamics of the spider silk fibers. The blocks of (Glu)4(Ala)6 and (Ala)6(Glu)4 at both ends formed AP- structure after low pH treatment, which was a model of the silks after spinning as mentioned above.52 On the other hand, many partial structures have been reported for the spider silk in the glands before spinning.63-69 The 47-mer peptides after freeze drying treatment can be proposed as a model of the spider dragline silks before spinning in the solid state. However, the conformational analysis of the Gly-rich motif can be slightly complicated because the blocks of (Glu)4(Ala)6 and (Ala)6(Glu)4 are expected to be a mixture of several conformations after the freeze drying treatment of the peptide. Yet, the conformations of (Glu)4(Ala)6 and (Ala)6(Glu)4 are expected to be mainly -helix after TFA treatment because poly-Ala motif ((Ala)12 or13) in S. c. ricini silk fibroin or their sequential model peptides was mainly -helix conformation after the treatment.70,71 Therefore, before analyzing the freeze dried samples, TFA treatment of the 47-mer peptide is useful in order to study the effect of the conformational change of the blocks of (Glu)4(Ala)6 and (Ala)6(Glu)4 that form -helices, which cause the conformational changes of each residue in the Gly-rich motif. The conformation of each residue in Gly-rich motif is then compared among TFA, low pH, and freeze drying treatments. Moreover, in order to use the spider dragline silks for applications, it is important to characterize the structural and dynamical changes in poly-Ala and Gly-rich motifs by changing the external environments. This information will help in the molecular design of the spider silk-based materials.72 The phenomenon of supercontraction of spider silk fibers in water has attracted many researchers for a long time.9-16 The change in the structure and dynamics of Gly-rich motif


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by hydration is undoubtedly a key to the origin of supercontraction.1-3,6,7 Thus, in connection with the origin of supercontraction, the conformational and dynamical change of the each 13C labeled residue in the Gly-rich motif was studied by hydration of the 47-mer peptide in the AP- poly-Ala motif. We showed earlier that the combination of the solid state NMR methods (13C refocused insensitive nuclei enhanced by polarization transfer (r-INEPT), dipolar decoupled (DD)/MAS and CP/MAS) provided useful information on this interesting phenomenon.26,27,29,30 The 13C r-INEPT observes the mobile components in the hydrated silk proteins with fast isotropic motion (>105 Hz). In contrast, 13C CP/MAS NMR observes the immobile components in the proteins or those with very slow motion (

Changes in the Local Structure of Nephila clavipes Dragline Silk

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