Sericin promotes Fibroin Silk I stabilization across a phase-separation

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Sericin promotes Fibroin Silk I stabilization across a phase-separation Hyo Won Kwak, Ji Eun Ju, Munju Shin, Chris Holland, and Ki Hoon Lee Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 13, 2017

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Biomacromolecules

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Sericin promotes Fibroin Silk I stabilization across

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a phase-separation

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Hyo Won Kwak1†, Ji Eun Ju1, Munju Shin1, Chris Holland2, and Ki Hoon Lee1,*

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University, Seoul 151-921, Korea

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3JD

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Keywords: Silk fibroin, Silk sericin, Phase-separation, Silk I structure, Gelation

Department of Biosystems & Biomaterials Science and Engineering, Seoul National

Department of Materials Science and Engineering, The University of Sheffield, Sheffield, S1

ACS Paragon Plus Environment

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Abstract: Natural silk spinning offers several advantages over the synthetic fiber spinning,

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although the underlying mechanisms of this process are yet to be fully elucidated. Silkworm

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silks, specifically which of B. mori, comprise of two main proteins; fibroin, which forms the

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fiber, and sericin, a co-extruded coating which acts as a matrix in the resulting non-woven

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composite cocoon. To date, most studies have focused on fibroins’ self-assembly and

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gelation, with the influence of sericin during spinning receiving little to no attention. This

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study investigates sericin’s effects on the self-assembly of fibroin via their natural phase-

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separation. Through changes in sample opacity, FTIR and XRD, we report that increasing

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sericin concentration retards the time to gelation and β-sheet formation of fibroin, causing it

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to adopt a Silk I conformation. Such findings have important implications for both the natural

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silk spinning process and any future industrial applications, suggesting that sericin may be

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able to induce long range conformational and stability control in silk fibroin whilst being in a

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separate phase a factor that would facilitate long term storage or silk feedstocks.

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Biomacromolecules

1. Introduction

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The mulberry silkworm, Bombyx mori silk has been widely used in industry due to its broad

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domestication, overall quality and more recently for use in high tech applications1–3.

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However, despite its utility, certain details of the natural spinning process represent a gap in

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our knowledge. When comparing the natural spinning process to industrial approaches, silk

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spinning appears to be a type of dry spinning, wherein a protein solution is moved through a

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silk gland, solidifies into a fiber and is then excreted4,5. This secreted fiber is used by the

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silkworm B. mori to form a protective cocoon prior to metamorphosis and is a continuous

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strand composed of two proteins, namely, fibroin and sericin. Fibroin constitutes 75% of the

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fiber strand weight and functions as the structural component, the remaining 25%, sericin, a

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group of polypeptides, has been assigned a minor role and is thought to be simply a protein

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that glues fibroin threads together during cocoon construction6,7.

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During the silk spinning process, silk dope solution undergoes changes in its composition and

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physical state8,9. For this reason, much attention has been focused on the physiological

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condition such as pH, ion concentration, temperature, and external forces on the self-

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assembly of fibroin10,11. However, recently, there is evidence to suggest there may be a

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further role for silk sericin in this process. Ki et al.12 reported the effect of sericin on the

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secondary structure of fibroin during wet spinning, and Hang et al.13 suggested some

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interaction between sericin and fibroin during co-axial electrospinning. However the most

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compelling evidence to date comes from Lee14 who observed micelle structures in a blended

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film of fibroin and sericin and speculated that the hydrogen bonds between fibroin and sericin

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retard the β-sheet crystallization of fibroin. However, whilst dry films are clearly different to

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the natural liquid state of the proteins found in the silk gland, this work does suggest that

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sericin may influence and perhaps even mediate the solidification kinetics and structure of

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fibroin.


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Therefore, the focus of this study is to mimic and understand the interaction between sericin

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and fibroin during storage as opposed to, the spinning. To this end, we have investigated the

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interaction between fibroin and sericin in solution. Using these protein’s natural propensity

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for macro phase-separation, we have been able to control the concentration of sericin and

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determine the impact it has on gelation and β-sheet conformational transition kinetics of

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fibroin.

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2. Materials and methods

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2.1. Materials

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Bombyx mori silkworm cocoons were kindly provided by National Academy of Agricultural

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Science. Formic acid, sodium carbonate (Na2CO3), sodium oleate, and calcium chloride

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(CaCl2) purchased from Sigma-Aldrich (USA). Lithium bromide (LiBr) was purchased from

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Acros (USA). Other reagents were purchased from Sigma Aldrich (USA).

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2.2. Methods

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2.2.1. Preparation of regenerated aqueous fibroin and sericin solution

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Silk cocoons were degummed twice with 0.02% (w/v) sodium carbonate and 0.03% (w/v)

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Marseille soap solution at 100 ℃ for 30 min, then rinsed with distilled water to remove

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residual sericin. Following this, the fibroin was dried in an oven at 50 ℃.

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To obtain high molecular weight, undegraded, sericin, the urea/mercaptoethanol extraction

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method was used on cocoons that had been cut into small pieces15. In brief, the cocoon pieces

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were immersed in a 5% (v/v) 2-mercaptoethanol in 8 M urea solution and heated to 80 ℃ for

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10 min, then the sericin containing solution was filtered through a nonwoven filter to remove

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any remaining pieces of cocoon. The solution was then dialyzed in distilled water using a

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cellulose membrane (molecular weight cut off = 6000–8000 Da, Spectrum Laboratories, Inc.)


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for two days to remove residual urea and mercaptoethanol. Finally, the extracted sericin

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solution was lyophilized and stored in desiccator prior to use.

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For the preparation of aqueous regenerated fibroin and sericin solutions, the solid extracted

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fibroin (10%, w/v) and sericin (2%, w/v) materials were dissolved in a 9.3 M LiBr solution

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separately at 50 ℃ for 4 h. The solution was then dialyzed in distilled water with a cellulose

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membrane for two days to remove the residual LiBr. To assess the removal of LiBr after

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dialysis, conductivity of the dialysate was checked every time until it had the same value as

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distilled water. The final fibroin and sericin concentration was adjusted 4%, 1% (w/v) with

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distilled water respectively.

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1.1.1. Gelation experiment of fibroin

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To monitor the gelation of fibroin with phase-separated sericin, 10 ml of aqueous fibroin

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solution added to an empty 20 ml vial. Then, 5 ml of distilled water and aqueous sericin (0,

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0.25, 0.50 and 1.00%, w/v) with 100 ppm of rhodamine B dye was carefully added above the

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fibroin solution using a syringe pump (KD Scientific, USA) in order to maintain the interface

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between the two solutions. Following this, the phase-separation fibroin and sericin samples

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incubated at 50°C were observed using digital camera until gelation had occurred in each

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fibroin solution.

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In a separate experiment, gelation kinetics were investigated using a microplate reader

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(Synergy H1, Biotek, USA) measuring sample turbidity changes at 550nm as a result of a

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heterogeneous microstructures causing an increase in the degree of light scattering. Using a

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24-well plate, per well, 2.0 mL of aqueous fibroin was added then, 1.0 mL of distilled water

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or aqueous sericin with various concentrations (0, 0.25, 0.50, and 1.00%, w/v) were added the

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above the fibroin solution (Fig. 1). As controls, bovine serum albumin (BSA, 1%, w/v) and

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polyethylene glycol (PEG 200K, 1%, w/v), were selected. Gelation time was defined as the


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time from the beginning of incubation at 50°C to the point when the plateau of optical density

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was reached 16.

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A fluorescence spectrum (FLS) was also recorded using the above microplate reader. For

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fluorescence measurements, 20 µM of thioflavin T (ThT) was added to the fibroin (4%, w/v)

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solution and black plates used (Vision plate 24, 4titude, United Kingdom). The excitation

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wavelength was set at 420 nm and the emission readings were taken at 480 nm. Time interval

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for the gelation kinetics was 1 h. Relative gelation kinetics was obtained by following as:

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ß-sheet transition kinetics (%) =

× 100

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IntensitySFg = Fluorescence intensity of fibroin when gelation completed

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IntensitySFs = Fluorescence intensity of fibroin solution at time0

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IntenstitySFt = Fluorescence intensity of fibroin solution at timet

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1.1.2. Characterization of fibroin solution and hydrogel

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1.1.2.1. Fourier-transform infrared (FTIR) spectroscopy

108

Fibroin solutions and hydrogels were lyophilized and turned into pellets (diameter < 13 mm)

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using a pellet press (The PIKE Technologies, USA) prior to ATR-FTIR characterization of

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the amide I band between 1600–1700 cm-1 (FTIR Nicolet iS5 with ATR accessory, Thermo

111

Scientific, USA). For each measurement, 128 scans were taken with a resolution of 4 cm-1.

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Aquired spectra were first subjected to a baseline correction using a straight line between

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1600 and 1700 cm-1 and then a nine-point multi-peak Gaussian fitting was performed using

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Origin software with structures assigned according to previous studies17–19 and the relative

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area of the single bands underneath the amide I region (Table. 1).:

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Table. 1. FTIR band Assignments in the Amide I Region for B. mori Silk Fibroin17–19.


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Wavenumber (cm-1)

Secondary structure

1605 -1615 1616 -1637 1638 -1655 1656 -1662 1663 -1696

Aggregated strands β-sheet Random coil α-helices β-turns

117 118

1.1.2.2.X-ray diffraction (XRD)

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After ATR-FTIR characterization, the same sample pellets were subjected to X-ray

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diffraction (XRD) using an X-ray diffractometer (D8 DISCOVER, Bruker, USA) with CuKa

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radiation. Irradiation conditions were 40 kV and 40 mA. XRD patterns were recorded at a

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speed of 2°/min at 40 kV and 35 mA in the region of 2θ from 5° to 40°.

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1.1.2.3. Mechanical tests

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To investigate the mechanical properties of fibroin hydrogels, a uniaxial compression

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experiment was performed. Cylindrical hydrogels with flat and parallel surfaces 15 mm

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diameter and 10 mm high were prepared and allowed to swell in distilled water for 24 h prior

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to testing. The samples were then removed from the water and subjected to compression tests

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at room temperature using a Universal Testing Machine (Lloyd Instruments, Ltd., United

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Kingdom) equipped with a 500 N load cell and a cross head speed of 10 mm/min. A total of 4

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hydrogels were tested for each treatment (N=4).

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1.1.2.4. Dissolution and enzymatic degradation test

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Lyophilized fibroin hydrogel samples weighing 15 mg (±1 mg) were incubated at 37 ℃ in

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phosphate-buffered saline (PBS) solution containing 1 U/ml α-chymotrypsin from human

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pancreas in 1.5 ml micro-centrifuge tubes. As a control, samples were also incubated in PBS

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at 37 ℃ without enzymes investigate the dissolution properties of fibroin in PBS. After one


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day of incubation, samples were centrifuged at 10,000 rpm for 10 minutes and the remaining

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silk pellet was washed twice with distilled water. After rinsing, fibroin residues were

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centrifuged again at 10,000 rpm for 10 minutes. The fibroin pellets were dried overnight in a

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fume hood and the mass determined using a 4-point analytical balance. Four samples from

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each group were taken at each time point (N= 4).

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2. Results and discussion

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To mimic the in vivo interactions between these silk proteins, we used fibroin and sericin’s

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natural tendency to phase-separate. During the silkworm’s 5th and final instar, fibroin is

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secreted into the posterior division silk gland and migrated into the middle division silk gland

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where sericin begins to be secreted20. From this point, both fibroin and sericin are stored as

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separate phases until spinning, and even after spinning they remain phase-separated21. Oh et

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al.22 found that the phase-separated state of fibroin and sericin could be maintained in vitro

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when the sericin solution was carefully loaded onto the top of the fibroin solution without

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disturbing the interface. Whilst the concentration of silk protein in the silk gland is around

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24%8, a regenerated sericin solution of more than 1% (w/v) easily converts into a gel state

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before the phase-separated gelation experiment is begun. For this reason, 1% (w/v) sericin

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was selected as the limit for this experiment and matched with a 4% (w/v) fibroin solution in

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order to maintain the natural ratio of sericin to fibroin as found in the fiber.

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In this study, rhodamine B dye, which has a strong affinity for fibroin, was used to monitor

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the interface between the two proteins during the gelation of fibroin23,24. Fig 1. shows the

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phase-separation state of fibroin (4.0%, w/v) and various concentrations of sericin (0, 0.25,

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0.5, 1.0%, w/v) alongside the gelation behavior of fibroin over time. During these tests the

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rhodamine B dye did not diffuse into the fibroin layer, indicating that the phase-separation

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between silk proteins was maintained in a liquid state until gelation (as seen by an increase in


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Biomacromolecules

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opacity of the fibroin phase). In addition to the use of an indicator dye, a control to check for

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protein migration across the layers was performed by testing for variations in dry weight

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concentration of fibroin and sericin layers before and after the gelation of fibroin (Fig. S1.).

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In all cases, variations in concentration before and after were negligible and within the

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standard deviation of the measurements, suggesting no significant migration. Comparing

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across samples, it was clear that the gelation of fibroin was delayed as the concentration of

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phase-separated sericin increased. This suggests that the sericin phase might play a

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significant role in the gelation kinetics of the fibroin solution.


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Figure 1. Typical experimental for the observation of gelation time of silk fibroin (SF) in a

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phase-separated with silk sericin (SS) above.


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This relationship was then investigated in more detail using an optical spectrometer. For

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comparison, other polymers, such as bovine serum albumin (BSA) and synthetic

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polyethylene glycol (PEG 200K), were selected as controls for the experiment (Fig 2.) Here

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the gelation time of the fibroin solution alone and with phases of distilled water, BSA or PEG

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200K was nearly 40 h. However, the gelation time of fibroin was nearly three times slower

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(115 h) when sericin was present in the upper layer, strongly indicating that sericin in

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particular can retard the gelation behavior of fibroin solutions.

(b) 120

(a) 3.5

SF SF + DW SF + PEG200K SF + BSA SF + SS

3.0

179

2.5

180

2.0

181

1.5

1.0

182

0.5

0.0 0

20

40

60

80

100

100

Gelation time (hrs)

178

O.D at 550nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

80

60

40

20

183120

0 SF

SF + DW

SF + PEG 200K

SF + BSA

SF + SS

Time (hrs)

184

Figure 2. Silk fibroin (SF) gelation delay with various polymers (1%) in the layer above SF

185

(SF at 4%). (a) Representative optical density changes and (b) gelation time of various SF

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solutions. The error bars represent the s.d. of the different observations (N=4).

187 188

To confirm observations from figure 2, to determine the effect of the sericin concentration in

189

the upper layer on the gelation of fibroin (4.0%, w/v), various sericin concentrations (0%,

190

0.25%, 0.50%, 1.00%, w/v) were prepared and tested (Fig. 3). Again, the fibroin gelation

191

time increased with increasing sericin concentrations (the effect of different concentrations of

192

fibroin may be found in figure S2). However, these results cannot completely exclude the

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possibility that fibroin and sericin mix when the concentration gradient or relative density is

194

not high enough to permit phase-separation. Interestingly, when fibroin and sericin were


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Biomacromolecules

195

mixed as a homogeneous solution, gelation did not occur until 160 h. These results imply that

196

sericin can clearly delay the gelation time of fibroin even though they are phase-separated.

197 198 (a) 4.0

SF SF + SS(0.25) SF + SS(1.00)

3.5

(b) 150

SF + DW SF + SS(0.50) SF + SS(1.00)Mix

199

120

200

Gelation time (hrs)

3.0

O.D at 550nm

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2.5 2.0 1.5

202

1.0

90

201 60

30

0.5

203 0.0

0

0

204

20

40

60

80

100

120

140

160

SF

SF + DW

SF + SS (0.25) SF + SS (0.50) SF + SS (1.00)

Time (hrs)

205

Figure 3. Silk fibroin (SF) gelation delay with various concentrations of silk sericin (SS) on

206

top of SF (4%, w/v), (a) representative optical density changes and (b) gelation time of

207

various SF solutions. The error bars represent the s.d. of the different observations (N=4).

208 209

Next we investigated the mechanism of fibroin gelation by using Thioflavin T (ThT), a

210

benzo-thiazole extrinsic fluorescence dye, which enables β-sheet formation to be

211

monitored25–28. Free ThT in an aqueous solution shows only weak fluorescence, with lower

212

excitation and emission maxima at 350 and 440 nm, respectively. However, when ThT

213

interacts with β-sheet-rich samples, it generates strong (red-shifted) fluorescence, with

214

excitation and emission maxima at approximately 440 and 490 nm, respectively29. Fig. 4(a)

215

shows that while the initial conformation of the fibroin solutions is mainly random coil, there

216

is some β-sheet content present and the intensity of fluorescence was increased in proportion

217

to the amount of fibroin. Fig. 4(b) shows the β-sheet transition kinetics of fibroin solution

218

(4%, w/v) in the presence of phase-separated sericin at different concentrations. This clearly


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219

indicates that the gelation of fibroin occurred with the conformational transition to β-

220

sheets16,30. The results agree well with previous studies and we can now associate an increase

221

in β-sheet content with gelation of fibroin. In addition, the transition into a β-sheet structure

222

was also retarded if the concentration of the sericin layer increased.

223 224 (a) 100000

SF (4.00) SF (2.00) SF (1.00) SF (0.50) DW

80000

226 227

60000

40000

20000

228

(b) 100

β−sheet transition kinetics (%)

225

Intensity (cps)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

80

60

40

SF SF + DW SF + SS (0.25) SF + SS (0.50) SF + SS (1.00)

20

0 0

229

Figure

460

480

500

520

540

560

580

600

0

10

Wavelengh (nm)

20

30

40

50

Time (hrs)

230

4. Relative β-sheet transition kinetics of silk fibroin (SF) based on Thioflavin T (ThT)

231

fluorescence intensity. (a) Fluorescence emission spectra of ThT in distilled water and in

232

various concentrations of SF solution (b) Relative β-sheet transition kinetics of SF (4% w/v)

233

based on ThT fluorescence intensity.

234

In order to obtain further structural information about fibroin gelation during the phase-

235

separated experiments, fibroin solutions were collected from the bottom layer of the sample

236

container after 1 h, lyophilized and subjected to ATR-FTIR and XRD analysis. Both tests

237

indicate no significant structural differences at the bottom layer between samples after 1 h

238

and confirmed a mainly random coil/an amorphous state of fibroin (Fig. S3).

239

However, post gelation, ATR-FTIR and XRD revealed significant differences between

240

samples (Fig. 5.). FTIR-ATR results show that as the concentration of the overlying sericin


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Biomacromolecules

241

phase increased, random coils and β-turn structures develop at the expense of β-sheets, α-

242

helices, and aggregated strands. XRD results provided more insight into this situation, with

243

data indicating that an increasing sericin concentration promotes a Silk I conformation of

244

fibroin to develop in the gel rather than the typical Silk II31,32.

245

Furthermore, if the sericin layer is removed post gelation and the fibroin gels allowed to stand

246

for 2 days, all samples converted to a Silk II structure (Fig. S4.) Thus it can be concluded that

247

phase-separated sericin retards fibroin Silk II formation by stabilizing Silk I structures, but

248

does not induce a permanent change in the gel.

249

SF gel post gelation with phase separated sericin (b)

(a) 100


250 80

252 253

Silk I : 11.9, 19.7, 24.7, 28.2 Silk II : 9.7,18.9

Relative intensity

251

Relative propotion (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60

40

20

0 SF

Turns

α-helix

SF + DW

SF + SS (0.25)

Random coil

SF + SS (0.50)

β-sheet

SF + SS (1.00)

254

Aggregated strands

10

Figure 5.

20

2 θ (degree)

30

254

40

Effect

255

of phase-separated silk sericin (SS) on the secondary structure (a) and XRD crystal properties

256

of silk fibroin (SF) gel (b).

257

Further evidence to support sericin’s ability to influence the properties of fibroin gels were

258

obtained by investigating fibroin’s mechanical properties and degradation profile. Fig. 6

259

shows the compressive strength and modulus of fibroin hydrogels prepared with different

260

upper phase concentrations of sericin. At the same fibroin concentrations, the mechanical

261

properties of the fibroin hydrogel were found to decrease with increasing concentrations of

262

sericin. This also coincides with changes in the secondary structure composition analysis by

263

FTIR and XRD. This is also consistent with other studies on hydrogel mechanical properties


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264

which have shown that a greater proportion of random coil structures may be responsible for

265

a weaker hydrogel33–35.

266 (b) 0.16

(a) 0.04

0.03

Compressive modulus (MPa)

267 Compressive strength (MPa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

268

0.02

269 0.01

0.12

0.08

0.04


270

0.00

0.00 SF

SF + DW

SF + SS (0.25)

SF + SS (0.50)

271

SF + SS (1.00)

25

30

35

40

45

Random coil proportion (%)

272

Figure 6.

Effect of phase-separated silk sericin (SS) concentration on the mechanical

273

properties of silk fibroin (SF) hydrogel. (a) compressive strength and (b) relationship between

274

random coil structure proportion and compressive modulus. The error bars represent the s.d.

275

of the different observations (N=4).

276

Since the random coil-rich, amorphous regions of fibroin are water soluble and degradable,

277

they are prone to both dissolution and proteolytic enzymes36 whilst the Silk II crystalline

278

region of fibroin isn’t

279

chymotrypsin were used to degrade fibroin. Fig. 7 and Fig. S5 shows that all fibroin

280

hydrogels exhibiting either Silk I or Silk II structures were insoluble in PBS. When subjected

281

to enzymatic degradation, fibroin hydrogels prepared with an upper phase-separation of

282

distilled water and 0.25% sericin solution had greater stability against enzymatic degradation,

283

which indicates Silk II structure formation. On the other hand, fibroin hydrogels prepared

284

with phase-separated 0.50% and 1.00% sericin could be enzymatically degraded, indicating

285

the existence of Silk I structures and backing up our XRD data.

37

. Therefore, after gelation and lyophilization, both PBS and α-

286


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Weight loss (%)

SF sol 1 h incubated with phase-separated sericin

(a) 20

PBS PBS + α−chymotrypsin

15 10 5 0

(b) 100

SF

SF + DW

SF + SS (0.25)

SF + SS (0.50)

SF + SS (1.00)

SF

SF + DW

SF + SS (0.25)

SF + SS (0.50)

SF + SS (1.00)

95

Weight loss (%)

SF gel post gelation with phase separated sericin

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90 85 80 75 70

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Figure 7. Water dissolution and enzymatic degradation properties of silk fibroin (SF) sol (a),

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SF gel phase-separated with different concentration of silk sericin (SS) (b). The error bars

290

represent the s.d. of the different observations (N=4).

291

Combined these results clearly show that the presence of a sericin layer induces the Silk I

292

structure in fibroin hydrogels. This interaction has to occur at the interface between sericin

293

and fibroin. Unfortunately, due to limited analysis techniques, it is difficult to identify

294

precisely the nature of the sericin and fibroin interaction. However, we can now begin to

295

speculate based our data and evidence from other reports. Lee14 has reported retardation of

296

fibroin crystallization in the presence of sericin in films. Ki et al.12 reported the effect of

297

sericin on the secondary structure of fibroin during wet spinning, and Hang et al.13 suggested

298

some interaction between sericin and fibroin during co-axial electrospinning. Their findings

299

indicate that sericin can affect the structural transition of fibroin at an interface. Based on

300

current and former results, the following mechanism is suggested for the development of Silk

301

I structures from fibroin in the presence of sericin. It has been reported that the gelation of

302

fibroin results from the conformational transition from random coil to β-sheet structures38.


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During this process, the hydrophobic part of fibroin, which consists of repeated sequences,

304

will start to agglomerate in order to avoid an unfavorable aqueous environment. The process

305

will continue until they find the most thermodynamically stable state, typically adopting a β-

306

sheet conformation (Silk II), which also acts as a physical cross-linker in the fibroin hydrogel.

307

However, we believe the interaction between fibroin and sericin at the interface may affect

308

the self-assembly of fibroin. This type of intervention by structure can be observed in other

309

self-assembling peptides. The Stupp group has studied the self-assembly of peptide-

310

amphiphiles and found that the β-sheet-stabilized self-assembly could only be found when the

311

sequence was maintained39. Therefore, even a slight disruption in the hydrogen-bonding

312

pattern of fibroin may prevent its β-sheet formation, which we propose to be caused by

313

sericin (Fig. 8). Here, this was seen by a promotion of Silk I structures in fibroin rather than

314

the usually more stable Silk II. This was then confirmed as upon removal of the sericin layer,

315

the Silk I structure of fibroin converted into Silk II.

316 317

Figure 8. Schematic of the effect of phase-separated silk sericin (SS) on the conformational


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transition of silk fibroin (SF).

319

From a biological perspective, the influence of sericin on fibroin may have profound effects.

320

Like the gelation observed in this study, natural spinning surrounds a conformational

321

transition of fibroin from a Silk I structure to a Silk II40. Therefore prior to spinning, the Silk I

322

structure is the typical structure of fibroin in the silk gland. Yet this has been difficult to

323

characterize in the past due to its inherent instability and tendency to convert into Silk II. Our

324

current results suggest that sericin may be responsible for inducing and maintaining the Silk I

325

structure in the animal prior to spinning, despite being phase-separated. Until now, its role

326

was underestimated as simply being a lubricant. However, the current results indicate that

327

sericin may have a further role in preventing premature fibroin crystallization in the gland.

328

3. Conclusions

329

Silk sericin may have more important roles than has been previously been recognized. In this

330

study, a phase-separated fibroin and sericin solution layer was constructed to mimic the

331

conditions inside in the middle of the silk gland. It was found that phase-separated sericin

332

slowed the gelation of fibroin, and this became more significant as the sericin concentration.

333

From Tht fluorescence intensity kinetics and ATR-FTIR measurements, this delay in gelation

334

correlated with slow β-sheet structure development in the fibroin layer with XRD indicating a

335

Silk I structure was promoted instead. Subsequent mechanical property tests and degradation

336

studies highlighted that not only structure, but other physical properties were influenced by

337

sericin. These results suggest that sericin, even though it was phase-separated, could prevent

338

premature crystallization of fibroin and induce a Silk I hydrogel structure. Interpreting these

339

findings in a wider context of the natural system, we propose that in the silk gland the sericin

340

layer that surrounds the fibroin acts to stabilize the metastable fibroin before spinning. These

341

findings could therefore have impact in the future storage and processing of fibroin and any


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biomimetic technological applications where careful control over the conversion and

343

solidification of fibroin is required.

344

ASSOCIATED CONTENT

345

Supporting Information

346

Effect of silk sericin (SS) concentration on the concentration changes of each bottom and

347

upper layer during the gelation of silk fibroin (SF). (Figure S1); Effect of silk sericin (SS)

348

(0.5%, w/v) on the increase in gelation time at different silk fibroin (SF) concentrations.

349

(Figure S2); Effect of phase-separated silk sericin (SS) on the secondary structure (a) and

350

XRD crystal properties of silk fibroin (SF) (b) during the initial 1 h incubation period. (Figure

351

S3); Effect of phase-separated silk sericin (SS) on the secondary structure (a) and XRD

352

crystal properties of silk fibroin (SF) (b) during further 48 h aging without upper sericin

353

phase. (Figure S4); Water dissolution and enzymatic degradation properties of silk fibroin

354

(SF) gel phase-separated with different concentration of silk sericin (SS) and further

355

incubation without upper layer for 48 h. (Figure S5).

356

AUTHOR INFORMATION

Corresponding Author

357 358

*Email: [email protected]

Present Addresses

359 360

†Department of Materials Science and Engineering, The University of Sheffield, Sheffield,

361

S1 3JD

362 363 364

Author Contributions


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The manuscript was written through contributions of all authors. All authors have given

366

approval to the final version of the manuscript.

Page 20 of 22

367

ACKNOWLEDGMENT

368

This work was supported by the Basic Science Research Program through the National

369

Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2010-

370

0025378 and NRF-2013R1A1A2059239) and the EPSRC, Project Reference EP/ K005693/1.

371

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Sericin promotes Fibroin Silk I stabilization across a phase-separation

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Hyo Won Kwak1†, Ji Eun Ju1, Munju Shin1, Chris Holland2, and Ki Hoon Lee1,*

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Sericin promotes Fibroin Silk I stabilization across a phase-separation

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