Article Cite This: Macromolecules XXXX, XXX, XXX-XXX
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NMR Investigation about Heterogeneous Structure and Dynamics of Recombinant Spider Silk in the Dry and Hydrated States Yugo Tasei,† Akio Nishimura,† Yu Suzuki,‡ Takehiro K. Sato,§ Junichi Sugahara,§ and Tetsuo Asakura*,† †
Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Nakacho, Koganei, Tokyo 184-8588, Japan Tenure-Track Program for Innovative Research, University of Fukui, 3-9-1 Bunkyo, Fukui-shi, Fukui 910-8507, Japan § Spiber Inc., 234-1 Mizukami, Kakuganji, Tsuruoka, Yamagata 997-0052, Japan ‡
S Supporting Information *
ABSTRACT: Spider silks continue to attract researchers because of their excellent mechanical properties and supercontraction behavior. In this paper, the structure and dynamics of recombinant spider silk protein (RSP) were characterized using 13C CP/MAS, 13C DD/MAS, and 13C refocused-INEPT NMR spectroscopies in the dry and hydrated states. The fractions of several structures of RSP with helical, random coil, and β-sheet polyalanine sequences were determined from the CP/MAS NMR spectra in the dry state. The CP/MAS NMR spectra changed to very simple one with dominant β-sheet Ala peaks by hydration due to a significant loss in CP signals of the other mobile carbons. On the contrary, only sharp mobile peaks, and both mobile and immobile peaks could be observed in the refocused-INEPT and DD/ MAS NMR spectra, respectively. The cis/trans proportion of the Gly−Pro bond was also determined. Our measurements provide new insight into understanding the supercontraction phenomenon of spider silks.
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important to characterize the fiber formation process and supercontraction behavior of the artificial silk fibers in detail. So far, the structure of the silk fibroins including spider silks has been studied using a wide variations of spectroscopic techniques including nuclear magnetic resonance (NMR),18,30−49 Raman spectroscopy,50−53 and X-ray diffraction (XRD) analyses.54−60 They have provided many insights into the molecular structure and dynamics of the silk proteins. However, a complete picture of the structure and dynamics in the molecular level of spider dragline silk is still unknown due to the complex and amorphous nature of the silk proteins in the dry and hydrated states. The most detailed molecular level picture of the structure and dynamics of silk has been obtained from NMR spectroscopy. The conformation-dependent NMR chemical shifts coupled with selective labeling have been used for determination of a local conformation in an amino-acidspecific sequence of silk.46,61−63 Other solid-state NMR techniques, that is, 2D spin-diffusion NMR,31,33,36,45 rotational echo double resonance (REDOR),32,41,44 13C−1H dipolar assisted rotational resonance (DARR),38,43,46,51 and so on have been used for structural determination of heterogeneous silk proteins in atomic resolution. In addition, for the characterization of silk structure in the hydrated state, combination of three solid-state NMR methods (13C refucused(r)-INEPT, DD/MAS, and CP/MAS) provides useful information.64−66 The r-INEPT where the pulse sequence was developed for solution NMR selectively observes the
INTRODUCTION Spider dragline silks possess extraordinary mechanical properties by combining high tensile strength with outstanding elongation before breaking that are superior to most man-made fibers.1−8 Thus, spider silks continue to attract attention of researchers in biology, biochemistry, biophysics, analytical chemistry, polymer technology, textile technology, and tissue engineering. All spider silks are made up of proteins named commonly spidroins. The major ampullate silk of the most studied spider genera Nephila, Argiope, Latrodectus, and Araneus consists of two proteins called major ampullated spidroin 1 (MaSp1) and spidroin 2 (MaSp2).9−12 These assemble into a fiber with the extraordinary mechanical properties. Another unique characteristic of the spider silks is supercontraction which occurs in the process of hydration. Interaction with water causes spider dragline silk fiber to contract up to 50% in length and swell in diameter.13−17 The Pro-related sequences have been emphasized to play a key role in the high elasticity and supercontraction of spider silk protein previously.16,17 Recent developments in biotechnology have opened the possibility to reproduce the recombinant spider silk proteins.18−23 The recombinant spider silk proteins which are produced by E. coli are generally obtained in powder form. Then, the silk powder dissolves in appropriate solvents for wet spinning.24 Commonly used organic solvent in preparation of silk fibers is hexafluoroisopropanol (HFIP) which allows for higher concentrations of silk protein dope. This solvent was widely used for preparation of spider dragline silk fiber as well as Bombyx mori silk fibroin fiber.12,15,21,24−29 In order to prepare the artificial silk fibers with excellent mechanical properties and promote the wide applications of the fibers, it is © XXXX American Chemical Society
Received: August 29, 2017 Revised: October 4, 2017
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DOI: 10.1021/acs.macromol.7b01862 Macromolecules XXXX, XXX, XXX−XXX
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Macromolecules
coli. and purified using a Ni column.10,11,21 The amino acid sequence of RSP prepared here is shown in Figure 1 where His-tag was attached to the N-terminal side of the amino acid sequence and used for the sample purification. [3-13C]Ala-labeled RSP sample was produced similarly by the cultivation in the medium containing [3-13C]Ala.69 Here [3-13C]Ala (each 99% enrichment) was purchased from Cambridge Isotope Laboratories, Inc., Andover, MA. These nonlabeled and 13C-labeled RSP samples in powder forms were fully immersed in methanol in order to convert the structures to β-sheet completely, which was named as MeOH-RSP powder here. The RSP sample dissolved in HFIP (Wako Pure Chemical Industries Ltd., Japan) was used for solution NMR observation. Then the RSP solution was dried for 10 days in vacuo at room temperature in the presence of phosphorus pentoxide to prepare the film, which was named as HFIP-RSP film. Small amounts of HFIP were still left in the film with α-helix form in the dry state as will be described later. The film was soluble in water only a short period of time, and we could prepare H2O-RSP film with random coil form in the dry state. In this process, HFIP was removed from the film. The stretched RSP fiber was prepared as follows.26−29 The HFIP solution of RSP with high concentration, 13.5% (w/w), was prepared as dope. The RSP as spun fibers were produced by extruding the dope solution from the nozzle (diameter 0.2 mm, length 1.2 mm) using high-pressure N2 gas into the methanol coagulation bath at room temperature through the air gap of 2 cm. The RSP fibers were soaked in the methanol bath overnight to remove HFIP from the fiber completely and then dried overnight. The fibers were then stretched to 3.5 times their original lengths using a manual drawing method in the air. The stretched SF fibers were dried overnight at room temperature under the fixed lengths. The fiber was named as MeOH-RSP fiber here. These RSP samples in the solid state were used for 13C solid-state NMR observation. 13 C and 1H Solution NMR Observations. The RSP powder was dissolved in either d 1 -HFIP ((CF 3 ) 2 CHOD) or d 2 -HFIP ((CF3)2CDOD) (10% w/v). All solution NMR experiments were performed on a JEOL Resonance ECA600II spectrometer at 298 K. The assignments of the 1H and 13C peaks to the residues were accomplished by 13C single pulse, TOCSY, NOESY, 15N HSQC, 13C HSQC, and 13C HMBC.70,71 The 1D 13C solution NMR spectrum was recorded under proton decoupling, 250 ppm spectral width, 85 000 scans, and relaxation delay of 2 s. TOCSY and NOESY spectra were recorded with 15 ppm spectral width in both the t1 and t2 dimensions, 512 and 4096 complex points in the t1 and t2 dimensions, eight scans, and a mixing time of 100 and 150 ms, respectively. 15N HSQC was recorded with 70 ppm spectral width in t1, 10 ppm in t2 dimensions, 256 and 2048 complex points in t1 and t2 dimensions, and eight scans. 13 C HSQC was recorded with 170 ppm spectral width in t1, 15 ppm in t2 dimension, 256 and 2048 complex points in t1 and t2, and 16 scans. 13 C HMBC was recorded with 250 ppm spectral width in t1, 15 ppm in t2 dimension, 256 and 2048 complex points in t1 and t2, respectively, and 128 scans were accumulated. The 13C chemical shifts of solution NMR were expressed by subtracting the value 2.5 ppm from the 13C chemical shifts represented as internal DSS reference for a comparison with the 13C solid-state NMR spectra of RSP.28,46,61−64 13 C Solid-State NMR Observation. The 13C CP/MAS NMR spectra of RSP powder, film and fiber in the dry and hydrated states were recorded using a Bruker Avance 400 NMR spectrometer with a 4 mm double resonance MAS probe and a MAS frequency of 8.5 kHz at room temperature. The RSP samples were carefully inserted into a zirconia rotor and sealed with PTFE insert to prevent dehydration of the hydrated samples during NMR measurement. Typical experimental parameters for the 13C CP/MAS NMR experiments were 3.5 μs 1H 90° pulse, 1 ms ramped CP pulse with 71.4 kHz rf field strength, TPPM 1H decoupling during acquisition, 2176 data points, 8K scans, and 4 s recycle delay. Details of the NMR conditions for the 13C DD/ MAS NMR experiments were described in our previous paper.68 A recycle delay of 5 s and 13C 90° pulse of 3.5 μs were used. Lorentz line broadening of 20 Hz was used for the 13C CP/MAS and DD/MAS NMR spectra. Typical experimental parameters for the 13C r-INEPT NMR experiments were 3.5 μs 1H and 3.5 μs 13C pulse, interpulse delay of 1/41JCH (1JCH = 145 Hz), refocusing delay of 1/31JCH, TPPM
mobile components of the hydrated silk proteins with fast isotropic motion (>105 Hz).67 In contrast, 13C CP/MAS NMR selectively observes the immobile components of the proteins or those with very slow motion (
