Strain Dependent Structural Changes in Major and Minor Ampullate

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Strain Dependent Structural Changes in Major and Minor Ampullate spider silk revealed by Two-Photon Excitation Polarization Irina Iachina, and Jonathan R. Brewer Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00368 • Publication Date (Web): 10 May 2019 Downloaded from http://pubs.acs.org on May 12, 2019

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Biomacromolecules

Strain Dependent Structural Changes in Major and Minor Ampullate spider silk revealed by Two-Photon Excitation Polarization ∗

Irina Iachina and Jonathan R. Brewer

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense

E-mail: [email protected]

Abstract Spider silk's mechanical properties make it an interesting material for many industrial applications. The structure and nanoscopic organization of its proteins are the basis of these qualities. In this study, the emission maxima of the auto-uorescence from the protein core from Major and Minor Ampullate silk bers from the orb-web weaving spider

Nephila Madagascariensis are determined and found to be 534 nm ± 11 nm

and 547 nm ± 19 nm. Molecular conformational changes during applied strain are observed in both ber types using two-photon excitation polarization measurements. Our ndings showed that within the bers the auto-uorescent dipoles are separated into two distinct populations, one randomly orientated (amorphous regions) and one with aligned dipoles as found in crystalline structures. The crystalline-amorphous ratio was determined and it was found that the crystalline dipoles made up around 30% and 20% of the auto-uorescent dipoles in Major and Minor Ampullate silk bers, respectively. Using two-photon polarization measurements it is possible to directly observe that the Major and Minor Ampullate silk bers structurally adapt to the applied

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stress, as well as discern dierent molecular conformational changes between Major and Minor Ampullates. It was seen that the crystalline-amorphous ratio increased, with up to 9% for Major bers and 6% for Minor bers, as strain was applied, suggesting a conformational adaptation of the ber, interpreted as non-crystalline 310 -helices transforming into crystalline β -sheets.

keywords: Major Ampullate silk, Minor Ampullate silk, Bombyx Mori, uorescence, two-

photon excitation polarization microscopy, strain dependency The properties of spider silk such as its strength, light weight, elasticity and that it is an environmentally friendly material made of proteins, has made it an interesting material for a large number of industrial applications. 1 Recently considerable interest has been given to the development of articial spider silk for applications in the elds of construction, transport and textile industries because it is nearly impossible to obtain industrial amounts of natural spider silk. 24 Much is already known about the structure of spider silk on both molecular and nanoscopic levels. 6,811 Nevertheless, the synthesized bers have not yet achieved the same properties as natural spider silk. 57 In order to produce articial silk with the same properties as spider silk, it is important to have a full understanding of the properties of natural spider silk. Spiders are able to produce several dierent types of silk, one of which is termed Major Ampullate silk (MAS), which is used for linking the spider to its web and is also known as dragline silk. 6 Another type of silk is the Minor Ampullate silk (MiS), which is used as a temporary scaold during the construction of the web. 11 Both ber types share some protein sequence motifs, but exhibit very dierent mechanical properties, investigation and comparison of the two bers can therefore give insight into how small structural changes on the molecular level give rise to signicant dierences in macroscopic properties.

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Biomacromolecules

Spider silk is thought to comprise a layered structure with an outer lipid layer (10-20 nm thick) surrounding several protein layers. 10 In MAS bers the innermost protein layer, referred to as the protein core (approx. 90% of the total thickness of the ber), contains polyalanine blocks forming highly ordered β sheets providing long-range order, resulting in nano-crystalline regions. Binding together the crystalline regions are glycine-rich blocks forming amorphous 310 -helices. 9,1215 The protein composition in the protein core of MiS bers resembles that of MAS bers but in MiS bers the crystalline regions are made up of alanine-glycine blocks and are separated from the amorphous region by spacers. 8,16 The macroscopic properties, such as the tensile strength and elasticity of spider silk is also very known. 57 The link between the macroscopic properties and the structural composition has been attempted to be answered through modeling, 1719 as well as using FT-IR spectroscopy, 2025 Raman spectroscopy, 26,27 and X-ray diraction. 25,27,28 Nevertheless, more information on the adaption of spider silk to stress on the molecular level is still needed to fully understand and utilize spider silk in biotechnical applications. Changes in uorescence polarization can be used to monitor organization of molecular dipoles. 2932 This method enables sensitive detection of the orientation of the excitation dipoles. Using two-photon excitation (TPE), the uorescence emission varies as cos4 to the angle between the electric eld of the excitation light and the excitation dipole of the molecule. 30,31 This results in TPE being much more sensitive to the molecular dipole orientation than single-photon excitation. TPE, compared to the other techniques mentioned above, also has a high spatial resolution in x,y and z (300 nm, 300 nm, 800 nm). The presence of auto uorescence from the spider silk proteins provides the basis for monitoring the orientation of their excitation dipoles and thus changes in molecular organization under 3



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deformation using two-photon excitation polarization (TPEP) measurements. Measurements were conducted on MAS and MiS bers, from the spider

, as a

Nephila Madagascariensis

function of the excitation light's polarization angle and applied strain. The TPEP measurements are used to couple the nanoscopic and macroscopic properties of the silk.

Materials & Methods Silk extraction Spider silk for all experiments was drawn from the spider

Nephila Madagascariensis

by

forceful silk extraction. Firstly, the spider was placed on a styrofoam surface and covered by a mesh. The mesh was then xed to the styrofoam surface with pins, thus immobilizing the spider. A hole is made in the mesh to allow access to the spinnerets. After having immobilized the spider, MAS bers were pulled out of the Major Ampullate gland by making contact with the spinneret with a tweezer and pulling gently. The silk ber was then fastened to a reeling machine using double-sided tape. MiS bers were reeled out with MAS bers but separated with tweezers in order to collect only MiS bers. The spider silk was reeled out at a constant speed of 7.7 mm·s-1 .

Excitation & Emission Excitation and single photon uorescence was measured on a Leica SP8 (Manheim, Germany). The excitation laser used was a pulsed white light laser. The system was equipped with a hybrid detector and used gated detection. Images were collected using galvo scanners. A 20X dry objective (NA=0.75) lens was used in measurements of single bers. The emission spectra were measured with an excitation wavelength of 475 nm. The emission spectra were recorded with time gating in order to remove all Raman scattering 4


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Biomacromolecules

produced by the crystalline regions within the protein core. Each emission spectrum for each ber was tted to a Gaussian distribution to determine the emission maximum. The average emission maxima for MAS and MiS silk bers were then determined.

Force pulley Single silk bers were mounted on a home-built force pulley that could be mounted on the microscope in order to image the sample during elongation. Upon elongation, a Miniature linear actuator, 60 mm travel (T-LA60A-S, Zaber Technologies Inc. Vancouver, Canada) pushes a TSB60M Translation stage, 60 mm travel (Zaber Technologies Inc. Vancouver, Canada) which in turn pulls on the end of the ber. The other end is xed to a UF1 isometric force sensor 0-50g (APPLIED MEASUREMENTS, Mercury House Calleva Park Aldermaston Berkshire UK), which measures the force exerted by the ber upon elongation. The force pulley is controlled via LabVIEW using an NI USB card NI USB-6211. The force pulley was then set to pull the ber at a constant speed of 0.05 mm/s.

Elasticity and tensile strength measurements In order to measure the elasticity and ultimate tensile strength of MAS and MiS bers the bers were mounted on the home-built force pulley mentioned above. Firstly the thickness of the ber was measured by imaging the auto-uorescence of the protein core in the two photon mode at 780 nm using a Ti:Sa laser (HPeMaiTai DeepSee, Spectra Physics, Mountain View, CA). It was decided to only use the thickness of the protein core as only this is thought to contribute to the mechanical properties of the bers. The laser power is controlled using motorized halfwave plate together with a polarizer. The objective used was a CFI S Plan Fluor ELWD 40X objective (NA=0.6) (Nikon). The force pulley was then set to pull the ber 20 mm at a constant speed of 0.05 mm/s. Measurements were stopped when the ber broke. The measurements were performed on 5 MAS bers and 10 MiS bers. 5



Two-photon excitation polarization In order to measure the change in uorescence signal as a function of the polarization angle of the excitation, the sample was imaged on a custom-built two-photon uorescence microscope as described in 31 (Figure S5). The sample is excited in the two-photon mode at 780 nm using a Ti:Sa laser (HPeMaiTai DeepSee, Spectra Physics, Mountain View, CA). The laser power is controlled using a motorized halfwave plate together with a polarizer. The objective used was a CFI S Plan Fluor ELWD 40X objective (NA=0.6) (Nikon). Before passing through the objective, the excitation light passes through a rotatable halfwave plate in order to control the polarization angle (φ) of the light. After each image is collected, the half-wave plate rotates ten degrees corresponding to a 20-degree rotation of the polarization of the light. The angle at which the light is polarized parallel and perpendicular to the stage is found. The sample is then imaged 19 times, corresponding to a complete rotation of 360 degrees of the excitation light. These images show the intensity of the auto-uorescence as a function of polarization angle of the excitation light. The resulting uorescence signal is passed through a tube lens and a 525 nm ± 25 nm bandpass lter (Semrock) before entering a Hamamatsu H7422P-40 photomultiplier. After acquiring intensity images of the auto-uorescence, the images were computed with SimFCS software. The sample was imaged at dierent strain values. For each strain value, the intensity of auto-uorescence as a function of polarization angle is measured at three dierent locations on a single ber. This is then repeated for ve dierent bers. For each ber, the mean auto-uorescence intensity per area is found for each strain value. The total mean autouorescence intensity is then found for all ve bers at each strain value. These are then plotted as a function of polarization angle of excitation light. 6


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Biomacromolecules

After each measurement, a reference sample of Rhodamine B in PBS buer was also measured in order to eliminated changes in intensity of the uorescence signal caused by existing uctuations in the system. To determine whether the amplitude changed for spider silk bers at dierent strain values, the obtained values of uorescence intensity as a function of polarization angle, φ of the excitation light for each ber at each strain value were tted with the following equation in RStudioTM , Boston, USA. A(sin4 (φ + b)) + c,

(1)

where A is the amplitude, b is the phase, and c is a constant. The values were tted to sin4 in order to have two peaks within the range of polarization angles. For each sample at each strain value, the amplitude was found with eq. 1 as well as the crystalline-amorphous ratio (CAR) which is dened by Eq. 2.

Results and discussion To characterize the bers Young's modulus (E) and the ultimate tensile strength (UTS) of the MAS and MiS bers were measured. For MAS bers, they were found to be: E=13.8 GPa ± 2.7 and UTS=1.2 GPa ± 0.2, and for MiS bers, they were: E=7.3 GPa ± 0.9 and UTS=537 MPa ± 71. The results show that the bers have dierent macroscopic properties. While MAS has the higher UTS, MiS has a lower Young's modulus, making it a more elastic ber. The protein cores in MAS and MiS bers were found to be auto-uorescent, enabling TPEP measurements. The emission spectra of MAS and MiS bers were recorded at their excitation maxima (475 nm) (Figure 1). The exact uorescent moiety of the protein remains to be identied. However, the proteins within the ber contain the amino acid Tyrosine(Tyr) and generally proteins with some amounts of Tyr exhibit some uorescence. Usually Tyr 7



exhibits uorescence in the UV spectrum, but this amino acid within a tertiary protein structure might have a shifted uorescence into the visible spectrum. 33,34 It has also been shown that non-conjugated systems can also exhibit uorescence in the visible spectrum by aggregation formation emission, which might also cause auto-uorescence within the spider silk proteins. 3537

Figure 1: Average emission spectra for MAS bers, N=4 (blue) and MiS bers, N=3 (red). Errorbars show the standard error. Excitation wavelength is 475 nm. Although considerable natural variation in the spectra of individual bers was found, the MAS bers were on average found to have an emission maximum at 534 nm ± 11 nm, which was lower than that found for MiS (547 nm ± 19 nm). In conclusion it was found that although the two bers had dierent emission maxima, it was not possible to be able to distinguish between the two ber types based solely on emission. Using the auto-uorescence from the ber core, it was possible to perform TPEP measurements to determine the orientation of the excitation dipoles of the proteins within the spider silk bers, as TPEP measurements can be used to discriminate between dierent types of molecular organization. Figure 2 shows modelled emission intensities as a function of the 8


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Biomacromolecules

polarization angle of the excitation light for randomly orientated excitation dipoles, Gaussian distributed excitation dipoles, crystalline (all excitation dipoles orientated in one direction) and a mix of crystalline and randomly orientated excitation dipoles.

Figure 2: Modelled emission intensities as a function of the angle of polarization of the two-photon excitation light, φ for dierent sample types. Black line: For a sample comprising only one excitation dipole or a crystal in which all excitation dipoles are arranged in the same direction, the intensity will depend on φ as cos 4 (φ + π/2). Red line: A sample consisting of multiple excitation dipoles orientated in a Gaussian distribution with a mean value at π /2 and a variance of 0.7 radians. Blue line: A mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos 4 (φ + π/2) + c. Green Line: a sample of randomly orientated excitation dipoles. Shown is the maximum uorescence intensity: M and the baseline: B. It is seen that randomly orientated excitation dipoles result in a constant intensity with no angle dependency. In a system comprising only one excitation dipole or a crystal in which all excitation dipoles are arranged in the same direction, the emission intensity will depend on the polarization angle of the excitation light, φ as cos 4 (φ). For a mixture of crystalline and randomly orientated excitation dipoles, a constant oset is observed compared to the 9



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crystalline case. For Gaussian distributed excitation dipoles, an increase of the full width half maximum (FWHM) of the peaks as well as a constant oset is observed. By tting with the above-mentioned models and analyzing the amplitude and FWHM of the peaks of TPEP data, it is possible to discern between the dierent types of molecular organization. 29

The crystalline amorphous ratio (CAR) which relates to the relative number of excitation dipoles orientated in a specic direction can be investigated by analyzing the amplitude and width of the peaks. The crystalline amorphous ratio (CAR) is dened as : CAR =

M −B , B·k+M −B

(2)

where M and B (Figure 2) are the maximum intensity and the baseline value respectively, and k is a correction factor, which considers the dierence in uorescence amplitude between amorphous and crystalline orientated emitters. The correction factor is needed because the maximum amplitude of several randomly orientated emitters will be lower than for the same number of crystalline emitters as the intensity of the random emitters is evenly spread over all angles while the intensity from crystalline emitters is collected to a combined maximum. This is found by calculating the ratio of intensities between a number of ordered and crystalline emitters. The emitted uorescence intensity measured as a function of the polarization angle of the excitation light can be seen in Figure 3. The TPEP measurements show the integrated intensity of many molecules/nanoscopic domains. Fluorescence intensity depends highly on the polarization angle of excitation light, showing that the excitation dipoles are not randomly orientated.

10


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Biomacromolecules

Figure 3: The auto-uorescence intensity variation of spider silk as a function of polarization angle of the excitation light (shown as white arrow). The angle in the images are from the top right: 1.02 rad, 1.36 rad, 2.38 rad, 2.72 rad, 3.06 rad and 3.4 rad. The ber is held at a strain value of 15%. It can be seen that the uorescence intensity varies with the polarization angle of the excitation light. The images are pseudo-coloured and the scalebar is 15 µm. In order to analyse the structural orientation of the excitation dipoles of the proteins in the silk, the intensity of the auto-uorescence as a function of polarization angle of the excitation light is measured. Further, to observe whether a change occurs upon strain this is measured for several dierent strain values. The experiment was performed on ve MAS bers (Figure 4A) and ve MiS bers (Figure 4B) and repeated at three dierent locations per ber per strain value.

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Figure 4: (A) Average uorescence intensity as a function of polarization angle of excitation light, φ [rad] for MAS bers at the following strain values: 0 (black), 0.10 (red), 0.15 (blue), 0.20 (green) and 0.25 (orange). The average uorescence intensity was found by measuring ve individual bers three times at each strain value. Errorbars show the standard error. (B) Example of tted data to the normalized uorescence intensity as a function of polarization angle of excitation light, φ [rad] for Strain = 0 (black). Model ts as described in Figure 2. Red line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.17. Red dashed line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.79. Blue line: A mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos 4 (φ) + c. For MAS bers, the measurements were performed using the following strain values: 0, 0.10, 0.15, 0.20 and 0.25. It was not possible to measure at higher strain values because the bers ruptured at strain values above 0.25. At a strain value of 0 (Figure 4A (black line)) the uorescence intensity was found to have two symmetrical maxima when the excitation light was polarized parallel to the ber length. Because the uorescence intensity is observed to be dependent on the polarization angle of the excitation light, it can be concluded that the excitation dipoles are not randomly orientated. Also, because the minimum intensity does not reach zero, the dipoles are not all orientated in one direction. The maximum intensity is achieved when the light is polarized parallel to the ber length, which shows that a large number of the excitation dipoles within the proteins are ordered 12


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Biomacromolecules

parallel to the spider silk ber. Fitting the data in Figure 4A to the models presented in Figure 2 results in Figure 4B (only shown for Strain = 0). Using a model consisting of Gaussian distributed excitation dipoles it was possible to t either the width of the peaks or the oset, but not both at the same time. First is shown the model describing a system consisting of Gaussian distributed excitation dipoles with a standard deviation, σ =0.17 (Figure 4B (red)). This model could t the width of the peaks, however could not t the oset of the data. Shown in (Figure 4B (red, dashed)) is a Gaussian distribution with a standard deviation, σ =0.79, and while the oset tted well, the width of the peaks is too wide. Lastly, the data was tted to the model describing a mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos 4 (φ) + c (Figure 4B (blue)). From this it seen that the excitation dipoles comprise two distinct populations of randomly and crystalline orientated dipoles and are not a Gaussian distribution of dipoles. Using the above conclusion for each strain value, the amplitude of the uorescence intensity peaks was found by tting the data to eq. 1 (Figure S1). The CAR was also found using eq. 2 and these values are summarized in Table 1. Table 1: Amplitude of the uorescence intensity peaks found by tting eq. 1, crystalline amorphous ratio (CAR) (eq. 2) and and p-value (p) of unpaired t-tests comparing all CAR values with the CAR value at strain = 0 for all MAS bers from Figure 4A (± standard error) Strain 0 0.10 0.15 0.20 0.25

Amplitude 1.39 ± 0.11 1.62 ± 0.19 1.62 ± 0.17 1.60 ± 0.16 1.32 ± 0.26

CAR p 0.32 ± 0.01 0.39 ± 0.02 0.006 0.38 ± 0.02 0.002 0.41 ± 0.01 < 0.001 0.39 ± 0.02 0.004

As mentioned earlier the proteins within the protein core assemble in several stacked β -sheets connected by amorphous 310 -helices. It has been shown that the moieties forming

both the β -sheets and 310 -helices are orientated parallel to the length of the ber. 23,26,38,39 The β -sheets form crystalline regions, with a high order parameter, 25 in the bers, 9,15 which 13



therefore suggests that the β -sheets make up the ordered part of the ber and are responsible for the ordered auto-uorescence. This is further supported by the CAR, which at strain = 0 was found to be 0.32 ± 0.01 suggesting that about 32 percent of the uorescent protein structures are highly ordered. This coincides with results observed with Raman spectroscopy and X-ray diraction, where it was found that MAS bers have a β -sheet content around 30%. 26,28 This also suggests that the 310 -helices are responsible for the auto-uorescence from the randomly orientated excitation dipoles. When stretching the bers to strain values 0.10 and 0.15 (Figure 4 A (red and blue line)), the symmetrical maxima are observed at the same angle but with an increased amplitude of 1.62. An increased amplitude could indicate an increased alignment of the ordered dipoles within the sample, meaning a reorientation of the existing molecular dipoles, or creation of new dipoles orientated in the same direction. If an increased alignment of the dipoles caused the increase in amplitude, this would be accompanied by a decreasing width of the peaks. The width of the peaks was examined by regarding the same data as Figure 4 A but with normalized intensities (Figure S2). No substantial changes in width were observed, indicating that changes in amplitude are not due to reorientation of the molecular dipoles. This is supported by X-ray diraction measurements as well as FT-IR spectroscopy, where no increased order of the β -sheets was observed upon applied stress. 22,28 The increased amplitude must therefore be caused by a formation of dipoles orientated in the direction of the ber length. This is further supported by the fact that the CAR increases from 0.32 ± 0.01 to 0.39 ± 0.02, meaning relatively more crystalline dipoles are formed upon elongation, suggesting that randomly orientated dipoles in the amorphous 310 helices might undergo a conformational change. Through modelling experiments, it has been shown that upon elongation at low strain values, the majority of the strain is located in the amorphous 310 -helices as these unravel rst. 18 Using IR measurements, it has been seen that upon stretching a MAS ber, the order parameter increases within the amorphous 310 helices as well as a change in conformation. 6,20,23,24 A conformational change of amorphous 14


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310 -helices transforming into more ordered/crystalline structures, such as β -sheets, could therefore explain the increased number of excitation dipoles in the direction of the ber length (Figure 5). Upon further elongation to strain values 0.20 (Figure 4 A (green line)) and 0.25 (Figure 4 A (orange line)), the amplitude decreases to 1.60 ± 0.16 and then to 1.32 ± 0.26, while the CAR remains constant. The decrease in amplitude follows the decrease in the number of emitters as calculated from the extension of the ber. It is also an interesting observation that within strain = 0 and 0.10 there is a substantial change in the CAR but from strain = 0.10 the CAR is constant. This suggests that the main change in reorientation of the molecules takes place within the linear strain range of the ber.

Figure 5: Schematic representation of proposed structural changes in MAS bers upon applied stress. Blue arrows represent stacked β -sheets that are connected by black 310 -helices. Upon applied force and elongation a conformational change occurs transforming 310 -helices into β -sheets. Results from TPEP experiments performed on MiS bers can be seen in Figure 6A. Similar to the MAS bers, the data suggest that the excitation dipoles of the proteins in the MiS bers consist of a mixture of randomly orientated and crystalline dipoles and not a Gaussian distribution of dipoles (Figure 6B).

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Figure 6: (A) Average uorescence intensity as a function of polarization angle of excitation light, φ [rad] for MiS bers at the following strain values: 0 (black), 0.10 (red), 0.15 (blue), 0.25 (green) and 0.35 (orange). The average uorescence intensity was found by measuring ve individual bers three times at each strain value. Errorbars show the standard error. (B) Example of tted data to the normalized uorescence intensity as a function of polarization angle of excitation light, φ [rad] for Strain = 0 (black). Model ts as described in Figure 2. Red line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.44. Red dashed line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.87. Blue line: A mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos 4 (φ) + c. The TPEP measurements were carried out for the following strain values: 0, 0.10, 0.15, 0.25 and 0.35. For each strain value, the amplitude of the uorescence intensity peaks was also found by tting the data to eq. 1 (Figure S3) and the percent of ordered dipoles was found using eq. 2.The measured amplitudes as well as the CAR values can be seen in Table 2. Table 2: Amplitude of the uorescence intensity peaks found by tting eq. 1, crystalline amorphous ratio (CAR) (eq. 2) and p-value (p) of unpaired t-tests comparing all CAR values with the CAR value at strain = 0 for all MiS bers from Figure 4 B (± standard error)

Strain 0 0.10 0.15 0.25 0.35

Amplitude CAR 0.30 ± 0.03 0.18 ± 0.02 0.36 ± 0.04 0.24 ± 0.02 0.37 ± 0.04 0.24 ± 0.02 0.37 ± 0.03 0.19 ± 0.008 0.40 ± 0.02 0.20 ± 0.004 16


p 0.02 0.03 0.81 0.28

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As seen with MAS bers, the amplitude of intensity of the auto-uorescence signal from the MiS bers increases and the width of the peaks does not change when the ber is strained to 0.10 and 0.15 (Figure S4) as well as an increase in CAR values. These observations are thought to be caused by the formation of crystalline dipoles orientated in the direction of the ber length, as in MAS bers. It is also noted that the CAR is generally lower than in MAS bers. This coincides with results from FT-IR experiments where it was found that the β -sheet content was lower in MiS bers than in MAS. 21 However, in contrast to the MAS bers, the CAR only exhibit an increase by maximum 6% upon elongation, while the maximum CAR increase for MAS bers was 9%. As mentioned earlier MiS bers contain spacers between the crystalline β -sheets and the 310 -helices. The stretching of these spacers, combined with a conformational change, could therefore explain the smaller increase in CAR. This might also explain the increased elasticity of these bers. As the amplitude and CAR increases upon applied stress it is thought that upon stretching a MiS ber two processes occur: 1. extension of the spacers between the β -sheets and 310 helices and 2. a change in conformation (Figure 7). 8 However, upon further strain to 0.25 and 0.35 an increase in width of the peaks is observed (Figure S4), which is also accompanied by a decrease in the CAR. This could be due to disordering of the crystalline units due to rupture of the brils within the ber. The overall uorescence was also seen to increase. The source of the increased uorescence is not yet understood, but could be due to a stress dependent uorescent molecule. This would need to be examined further.

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Figure 7: Schematic representation of proposed structural changes in MiS bers upon applied stress. Blue arrows represent stacked β -sheets that are connected by black 310 -helices and these are separated by green spacers. Upon applied force and elongation a conformational change occurs transforming 310 -helices into β -sheets, as well as stretching of the spacers. As TPEP utilizes the inherent uorescence in the sample it is not only limited to spider silk bers. Silk from Bombyx Mori has also been shown to be auto-uorescent and it is therefore also possible to analyse the orientation of the excitation dipoles and amount of order using TPEP for these ber types (Figure S6). This shows that TPEP can be used for various samples in order to analyse whether the samples are isotropic and to which degree.

Conclusion In conclusion it was found that within the ber 30% and 20% of the excitation dipoles are orientated along the length of the ber for MAS and MiS bers respectively. It was seen that the crystalline-amorphous ratio increased, with up to 9% for MAS bers and 6% for MiS bers, as strain was applied, suggesting a conformational change occurring within the protein motifs. The results show that both ber types are able to structurally adapt to the applied stress, which is important to consider when designing articial silk. The measurements also suggest that the molecular dierence between Major Ampullate and Minor Ampullate silk 18


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bers result in distinct conformational reactions to applied stress on a nanoscopic level, which in turn are reected in dierent macroscopic properties of the bers.

Supporting Information Fig. S1: Fluorescence intensity as a function of polarization angle of excitation light, φ [rad] for MAS bers, as well as the ts created using eq. 1. Fig. S2: Normalized uorescence intensity as a function of polarization angle of excitation light, φ [rad] for MAS bers. Fig. S3: Fluorescence intensity as a function of polarization angle of excitation light, φ [rad] for MiS bers, as well as the ts created using eq. 1. Fig. S4: Normalized uorescence intensity as a function of polarization angle of excitation light, φ [rad] for MiS bers. Fig. S5: Microscopy set-up for polarization measurements. Fig. S6: Normalized uorescence intensity as a function of polarization angle of excitation light, φ [rad] for Bombyx Mori bers.

Funding Sources Irina Iachina received funding for equipment from Siemensfonden Grant 11.

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Average emission spectra for MAS fibers, N=4 (blue) and MiS fibers, N=3 (red). Errorbars show the standard error. Excitation wavelength is 475 nm.


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Modelled emission intensities as a function of the angle of polarization of the two-photon excitation light, φ for different sample types. Black line: For a sample comprising only one excitation dipole or a crystal in which all excitation dipoles are arranged in the same direction, the intensity will depend on φ as cos4(φ+π/2). Red line: A sample consisting of multiple excitation dipoles orientated in a Gaussian distribution with a mean value at π/2 and a variance of 0.7 radians. Blue line: A mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos4(φ+π/2) +c. Green Line: a sample of randomly orientated excitation dipoles. Shown is the maximum fluorescence intensity: M and the baseline: B.



The auto-fluorescence intensity variation of spider silk as a function of polarization angle of the excitation light (shown as white arrow). The fiber is held at a strain value of 15%. It can be seen that the fluorescence intensity varies with the polarization angle of the excitation light. The images are pseudo-coloured and the scalebar is 15 μm.


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(A) Average fluorescence intensity as a function of polarization angle of excitation light, φ [rad] for MAS fibers at the following strain values: 0 (black), 0.10 (red), 0.15 (blue), 0.20 (green) and 0.25 (orange). The average fluorescence intensity was found by measuring five individual fibers three times at each strain value. Errorbars show the standard error. (B) Example of fitted data to the normalized fluorescence intensity as a function of polarization angle of excitation light, φ [rad] for Strain = 0 (black). Model fits as described in Figure 2. Red line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.17. Red dashed line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.79. Blue line: A mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos4(φ+π/2) +c.



Schematic representation of proposed structural changes in MAS fibers upon applied stress. Blue arrows represent stacked β-sheets that are connected by black 310-helices. Upon applied force and elongation a conformational change occurs transforming 310-helices into β-sheets.


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(A) Average fluorescence intensity as a function of polarization angle of excitation light, φ [rad] for MiS fibers at the following strain values: 0 (black), 0.10 (red), 0.15 (blue), 0.25 (green) and 0.35 (orange). The average fluorescence intensity was found by measuring five individual fibers three times at each strain value. Errorbars show the standard error. (B) Example of fitted data to the normalized fluorescence intensity as a function of polarization angle of excitation light, φ [rad] for Strain = 0 (black). Model fits as described in Figure 2. Red line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.44. Red dashed line: Excitation dipoles oriented in a Gaussian distribution with a variance of 0.87. Blue line: A mix of crystalline and randomly orientated excitation dipoles which depend on φ as cos4(φ+π/2) +c.



Schematic representation of proposed structural changes in MiS fibers upon applied stress. Blue arrows represent stacked β-sheets that are connected by black 310-helices and these are separated by green spacers. Upon applied force and elongation a conformational change occurs transforming 310-helices into βsheets, as well as stretching of the spacers.


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Strain Dependent Structural Changes in Major and Minor Ampullate

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