In Situ Identification of Secondary Structures in Unpurified Bombyx mori Silk Fibrils Using Polarized Two-Dimensional Infrared Spectroscopy

The mechanical properties of biomaterials are dictated by the interactions and conformations of their building blocks, typically proteins. Although the macroscopic behavior of biomaterials is widely studied, our understanding of the underlying molecular properties is generally limited. Among the noninvasive and label-free methods to investigate molecular structures, infrared spectroscopy is one of the most commonly used tools because the absorption bands of amide groups strongly depend on protein secondary structure. However, spectral congestion usually complicates the analysis of the amide spectrum. Here, we apply polarized two-dimensional (2D) infrared spectroscopy (IR) to directly identify the protein secondary structures in native silk films cast from Bombyx mori silk feedstock. Without any additional peak fitting, we find that the initial effect of hydration is an increase of the random coil content at the expense of the helical content, while the β-sheet content is unchanged and only increases at a later stage. This paper demonstrates that 2D-IR can be a valuable tool for characterizing biomaterials.

FTIR spectra of the hydrated silkworm films that are able to isotopically exhange H with D during the hydration process are those that are water accessible. Upon H/D exchange, the amide II shifts to lower frequency, absorbing around 1450 cm −1 and we refer to it as amide II'. The area of the vibrational band of the amide II' hence reflects the amount of groups in the film that can interact with water. Fig.   S2 shows the amide II' and amide II vibrational bands for Sample A discussed in the main text: from the calculated areas we can estimate that 70% of the NH groups are H/D exchanged to ND. Figure S2: Fit of the linear spectrum of hydrated silk film in the amide II region. To estimate the amount of amide groups that isotopically exchange (i.e. that are water accessible), we extract the areas of vibrational bands of the amide II', and amide II. To obtain a quantitatively good fit, we use three Gaussian-shaped peaks to describe the amide II absorption bands at 1450 cm -1 , and 2 Gaussian-shaped peaks to described the amide II at 1550 cm -1 .

Anisotropy
To obtain information over the molecular orientation, we calculate the anisotropy, defined as R = ∆αpar−∆αper ∆αpar+2∆αper , where ∆α par and ∆α per are the transient absorption changes measured in parallel and in perpendicular polarization configuration, respectively. In case of the diagonal peaks, the ratio between parallel and perpendicular signal is expected to be 3, leading to an anisotropy of 0.4. In Fig.S3, we report the anisotropy as a function of probe frequency obtained by centering the excitation pulse at the beta-sheet A ⊥ vibrational mode, which absorbs at 1623 cm -1 . We observe that at the probe frequency of the same mode, where the bleach of the diagonal peak is found in the 2DIR spectrum, the anisotropy value is perfectly at 0.4, indicating that the scaling factor between parallel and perpendicular is 3 in our experiments. We also observe that at 1710 cm -1 , where the β-sheet A vibrational mode absorbs, the anisotropy changes to negative values, approaching -0.2. In this case, the anisotropy represents the relative orientation of the A ⊥ transition dipole moment with respect to the A dipole moment. We can calculate the relative angle θ = arccos 5R 0 +1 3 , obtaining an angle of around 90 • , which is expected since A ⊥ and A have transition dipole moments that lie perpendicular to each other.

Absence of β-sheet secondary structures in untreated films
To confirm that the helical structure is the most abundant structure in untreated silk films, we measured another film that was prepared with the same protocol using a native silk feedstock liquid extracted from a different gland. Fig. S4 shows the "diagonal-free" spectrum this silk film before being hydrated. We here observe only the off-diagonal signatures of the helical structure, confirming that in silkworm films proteins adopt mostly helical conformation at ambient conditions.

Anti-diagonal slices
In Fig.S5 we report the diagonal free 2DIR spectra and the anti-diagonal slices from where we obtain the 2DIR signals reported in the main text. Figure S4: Subtracted 2D-IR spectrum of an untreated silk film, coming from a different batch than the sample discussed in the main text. The spectrum displays the cross-peaks associated to helical secondary structures (see white circles), while the cross-peaks signatures expected for β-sheet, which would be expected at the frequencies highlighted by the blue circles, are absent. Effect of exposure time to high humidity on β-sheet content The experimental data reported in this section refers to a sample from the same batch as the one shown in the main text and were measured at the University of Aarhus using a 10 kHz commercial time-domain 2DIR spectrometer (PhaseTech 2DQuickIR) described previously. 1,2 Briefly, femtosecond mid-IR pulses were split into pump and probe pulses.
The pump pulses were split into time-and phase-controlled pulse pairs using an acoustooptic pulse shaper, and then focused into the sample 500 fs before the probe pulse, which was dispersed onto an MCT detector (PhaseTech JackHammer). The delay between the pump pulse varied in 33 fs steps from 0 to 3 ps, and the measurements were performed in a 1300 cm-1 rotating frame. In order to reduce interference from scattered light, a 4-frame phase cycling scheme was used, and reference spectra recorded at -10 ps were subtracted.
The sample here analyzed is taken from the same silkworm film on which the experiments Figure S6: Anti-diagonal slice of the diagonal free spectrum measured from a silkworm film from the same batch as in the main text at different level of hydration: untreated (red solid line), hydrated for 2 hours (cyan solid line) and for 36 hours (blue solid line). The black boxes highlight the changes occurring in the cross-peaks associated to β-sheet secondary structures. The gray rectangle shadows the helical region, close to the main diagonal, that is affected by scattering in the shown measurement.
reported in the main text were performed. After exposing the film to saturated D 2 O (85% RH) no major change is observed in magnitude of the cross-peaks between the β-sheet modes at 1630 cm -1 , and at 1700 cm -1 , indicating that the β-sheet content is constant. This confirms and well-reproduces the result presented in the main text. We further expose this sample to high humidity for additional 34 hours. Upon this, the cross-peaks between the β-sheet modes change: the magnitude of the cross-peak at 1620 cm -1 increases drastically, while the cross-peak at 1700 cm -1 spits in two distinct subbands. This last effect is due to the overlap of different cross-peaks, likely associated to the hydrated and not-hydrated β-sheet. Because of the overlap, the relative intensities of the cross-peaks cancel partially off, and thus we do not consider it in our analysis. However, it is clearly visible that the magnitude of the cross-peak at 1620 cm -1 increases drastically, indicating that β-sheet structures are being formed after long exposure time (see blue curve in Fig. S6). In this section, we reports additional analysis of the linear infrared spectra shown in Fig.   1 of the manuscript as further consistency check of the results obtained from the 2D-IR measurements . Fig. S7-(a,b) shows the second derivative of the spectra of the untreated and hydrated silk films, respectively. It is possible to observe two significant minima around 1626 cm −1 and 1656 cm −1 , which are the values provided by the analysis of the 2D-IR spectra.

Multi-peak fitting
The linear absorption spectra (Fig. 1

Measurement reproducibility
In this section, we report the full analysis of the 2D-IR data collected on another silk worm film, from the same batch of the one analyzed in the main text. These additional data were collected at the University of Aarhus (see also Fig. S6) using 0.5 ps delay between the pump and probe pulse. The sample was hydrated using the same procedure reported in the main text. Fig. S10 reports the perpendicular (a,b) , diagonal-free (c,d) and cross-peak free (d,e) spectra of the untreated and partially hydrated film, respectively. It is possible to observe that the main results presented in the manuscript are nicely reproduced. In particular, it is possible to observe in Fig.S11 that upon partial hydration the β-sheet content does not increase, as also indicated by the lack of major modifications in the anti-diagonal slices (Fig.   S6). On the contrary there is an strong increase in the diagonal slice (see Fig. S11) around 1650 cm −1 , compatible with random-coil secondary structures. This feature is more visible in this second dataset because the data were collected at a shorter time (0.5 ps), where the contribution of the random coil to the bleach is expected to be stronger because the random coil has a shorter vibrational lifetime than the β-sheet. (e) (f) Figure S10: Perpendicular (a,b), diagonal-free (c,d) and cross-peak free spectra (e,f) measured with a pump-probe delay at 0.5 ps for an untreated and hydrated silkworm film. The data were collected at the University of Aarhus using a pump-probe delay of 0.5 ps. Figure S11: Untreated (red solid line) and treated (blue solid line) diagonal slices of the cross-peak free spectra shown in Fig. S10-(e,f).