Determining the Orientation of Proteins in Single Silk Fibers

Polarized attenuated reflection or ATR is an effective technique to determine the orientation of a large number of samples but more difficult to apply to really small samples such as silk fibers.

The golden gate single-reflection ATR accessory, which makes use of diamond as an ATR element and a focalized beam is highly effective to study quantitatively the conformation and orientation of a single silk fibroin filament of the silkworm Bombyx mori that is about 10μm in diameter.

Three methods are used to determine molecular orientation by ATR infrared spectroscopy. The most common one is to keep the sample fixed on ATR and rotate the electric field of the incident radiation. This method can be implemented as the sample does not have to be moved, but it requires complete knowledge of the components of the electric field in every direction at the surface of the ATR crystal.

Sample Preparation

Cocoons from the silkworm B. mori were obtained from the Insects Production Unit of the Canadian Forest Service (Sault Ste. Marie, Ontario, Canada). Raw B. mori silk comprises two fibroin filaments held together by a cementing layer of the protein sericin. In order to remove the sericin layer, degumming of cocoon silk was done in boiling water containing sodium bicarbonate (0.05% w/v) for 15min.

In order to remove the excess water, the resulting fibroin filaments were rinsed thoroughly with deionized water and dried under vacuum for a few minutes. Storage of the samples was done at 22 ± 1 °C and 65 ± 5% relative humidity.

Figure 1. Coordinate system and mechanical setup used for the sample rotation and dichroism measurements showing the angular wheel (A), the support bridge (B),the shaft (Z) and the elevation stopper (F).

Mechanical Setup for Sample Mounting and Rotation

For a range of angles between the electric field of the incident radiation and the fiber axis by rotating the sample polarized ATR spectra was recorded. The Golden Gate ATR accessory shaft was modified to accommodate the sample holder shown in Figure 1 to repeatedly have an almost identical contact between the sample and the ATR element for all spectra.

The sample holder includes an anvil formed by a barium fluoride (BaF₂) disk attached to an identical diameter plastic holder with double sided adhesive tape. Silk fibers were positioned at the center of the BaF₂ substrate and gently attached at both ends with adhesive tape on the side of the plastic holder by applying very little tension on the fiber keeping it straight and avoiding shrinking of the fiber.

A circular flange was used to attach the sample holder to the shaft of the bridge (B) of the ATR accessory. Between the aluminium flange and the plastic holder, a piece of soft low-density polyurethane foam was inserted in order to ensure the application of reproducible low pressure on the sample.

Spectral Acquisition and Treatment

A Nicolet Magna 850 Fourier transform infrared spectrometer was used to record the spectra with a liquid nitrogen cooled narrow-band mercury cadmium telluride (MCT) detector and a Golden Gate ATR accessory. The infrared beam in this apparatus is focused to a 750µm diameter on the diamond crystal with ZnSe lenses (4X magnification). Perpendicular to the plane of incidence the electrical field was polarized using a ZnSe wire-grid polarizer. From 128 scans at a resolution of 4cm^-1, each spectrum was obtained using a Happ-Genzel apodization.

The correction of all spectra was done for wavelength dependency of the of the penetration depth of the infrared radiation. The band fitting iteration routine used in GRAMS is based on a nonlinear algorithm known as the Levenberg-Marquardt method.

Results and Discussion

Figure 2 shows the ATR spectra between 950 and 1750 cm^-1 of a single fibroin filament of degummed B. mori cocoon silk obtained with the s-polarized light parallel at different rotation angles of the fiber from 0° (fiber parallel to the electric field) to 90° (fiber perpendicular to the electric field).

Figure 2. ATR spectra of a single B. mori silk fiber obtained with s-polarized light at different rotation angles of the fiber from 0° (fiber parallel to the electric field) to 90° (fiber perpendicular to the electric field).

The assignment of the major bands of B. mori silk is given in Table 1.

Table1. Assignment and orientation dependence of the observed bands in the polarized ATR spectra of B. mori silk.

^a Abbreviations used: v, stretching; d, bending; w, wagging; r, rocking; as, asymmetric; s, symmetric.

Figure 3 shows typical decomposed spectra for a fiber aligned at 0° and 90°. These spectra clearly confirm that the β-sheets are highly oriented in B. mori silk since the 1615 and 1698 cm^-1 components almost completely disappear in the 0° and 90° spectra, respectively.

Figure 3. Spectral decomposition of the experimental spectra of a single B. mori silkfiber recorded at 0° and 90°

Figure 4 shows the experimental absorbances of the components at the 1615, 1649, and 1698 cm^-1 plotted as a function of θ.

Figure 4. Integrated absorbance of the 1615, 1649, and 1698 cm^-1 band components as a function of the angle θ between the fiber axis and the electric field of the infrared radiation. The correlation with Eq. 1 is also shown.

For a specific vibration absorbance is proportional to the square of the scalar product between the transition moment (M) and the electric field of the infrared radiation (E), the absorbance should follow a cosine square function of the angle, θ between the fiber axis components such that:

where A_‖ and A_⊥ are absorbances measured when the infrared radiation is polarized parallel (0°) and perpendicular (90°) to the fiber axis, respectively, and δ is a phase factor that accounts for the error in the angular positioning of the fiber.

Equation 2 is used to obtain the dichroic ratio R allowing the calculation of the second moment P₂ (cosγ)or P₂ of the orientation distribution function expressed as a sum of Legendre polynomials in cos γ where γ is the angle between M and E as seen in Equation 3.

Table 2 provides the values of R and (< P₂ >) determined for the 1615, 1649 and 1698 cm^-1 bands.

Table 2. Polarized absorbances (A and A), dichroic ration(R), order parameter (< P₂ >) and relative area of the major components of the amide I band obtained from the fit of Equation 1.

In order to obtain more insights into the conformation of the silk fibroins, an orientation-independent or structural absorbance spectrum (A₀) can be calculated from the parallel and perpendicular polarized spectra using Equation 4.

Figure 5 shows the A_‖ and A_⊥ spectra of five different cocoon monofilaments.

Figure 5. Normalized spectra of five different B. mori silk fibers recorded at 0° and 90°.

The structural spectrum recorded at 54.7° and the one calculated from A_‖ (0°) and A_⊥ (90°) of Fig. 2 are shown in Figure 6.

Figure 6. ATR spectrum of a single fiber recorded at 55° and the orientation-independent spectrum A₀ calculated from the 0° and 90° spectra.

Conclusion

This work provides a convenient approach for routine analysis of small single fibers that can overcome the experimental limitations of TR and transmission experiments. perfectly reproducible, which can be achieved by using the sample holder developed in our laboratory. Even though two spectra are sufficient to calculate the orientation order parameter and the isotropic spectrum, the robustness of the reproducibility can be ascertained from measurements at different angles as described in this study. Multiple angle measurements are also beneficial for validating the band-fitting procedure, which is a necessary step to extract quantitative information relative to the different vibrational amide I modes.

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