3.1 Optimization of the probe
Fluorescence spectroscopy is widely used to interpret protein structure at atomic resolution. Aromatic amino acids, e.g., Trp, Tyr, and phenylalanine, offer intrinsic fluorescent probes for monitoring protein conformations, dynamics, and intermolecular interactions. Among them, Trp is the most popular probe for investigating protein structural changes because it is sensitive to the environment[20]. The fluorescence spectroscopy of Trp in SF was evaluated under different pH conditions to indicate changes in SF conformation. However, as shown in Fig. S1, the emission spectrum barely changed with pH. This result may be attributed to the fact that Trp is more sensitive to the water content of SF because most Trp residues are distributed on the surface of proteins, a property that induces poor sensitivity in aqueous solution. Thus, the external probe might be more suitable for our system.
ThT is perhaps the most widely used amyloid dye as this reagent specifically binds to the β-sheet structure of fibrils[21,22]. In this study, ThT was used as an external probe to monitor the conformational state of SF. As shown in Fig. 1a, the fluorescence intensity of ThT at 485 nm substantially increased upon binding to the hydrophobic region of SF compared with free ThT. Thus, this phenomenon was qualified for fluorescence assay. The relationship between ThT concentration and fluorescence intensities at 485 nm of the SF solution is depicted in Fig. 1b. The highest intensity was obtained with 20 µM ThT, and it decreased when the concentration exceeded 30µM probably because of the self-quenching effect of excessive ThT. Hence, 20 µM ThT was chosen as the optimum concentration and used for further analysis.
3.2 Effect of pH on structural changes in SF
pH is clearly an important factor in the formation of supramolecular assemblies of Bombyx mori SF because it has an influence on electrostatic interactions[12]. The fluorescence emission spectra of SF at pH 4.8, 5.0, 5.2, 5.6, and 6.8 are given in Fig. 2. These pH levels represent the conditions at different parts of the silk gland of Bombyx mori: pH 4.8 for the anterior division and pH 6.8 for the posterior division[23].
Emission intensity increased by about 3.6-fold when pH decreased from 6.8 to 4.8, indicating the β-sheet conformation of SF in aqueous solution. Photographs of SF solutions with the ThT probe at pH 4.8, 5.2, and 6.8 displayed the same trend. The fluorescence strategy could be used as a real-time method for monitoring the assembly process. The emission intensities at 485 nm initially increased with incubation time at pH 4.8 and 5.0 within 5 days and then decreased over time. This result might be ascribed to the aggregated formation and the self-quenching of the ThT dye. By contrast, the intensity scarcely increased at pH 6.8. The pH and time dependence of ThT fluorescence was also determined. Results showed that the effect of pH and incubation time were negligible for pure ThT (Fig. S2).
ANS is also a frequently used extrinsic fluorescence probe for exploring changes in hydrophobic regions during conformational transitions of SF. Its fluorescence behavior upon binding to SF was also investigated for comparison. The fluorescence spectrum of pure ANS exhibited a wide band with very low fluorescence intensity and an emission maximum of 542 nm in polar environments (Fig. S3a). The fluorescence intensity of ANS evidently increased in the presence of SF, which was accompanied by shifting of the emission maximum to 471 nm. The emission intensity of ANS at 470 nm also increased when pH decreased from 6.8 to 4.8. However, compared with ANS, the ThT probe showed better sensitivity as the fluorescence enhancement was more obvious. Compared with free ThT, the fluorescence of the SF solution with the ThT probe increased by about 25-fold at pH 4.8, whereas the fluorescence of the SF solution with the ANS probe increased by about 7-fold only (Fig. S3b). The difference in this phenomenon might be ascribed to the possibility that ANS was sensitive to the hydrophobic environment, whereas ThT was bound to the β-sheet structure. Moreover, SF (PI~4.5) is negatively charged at pH 4.8 to 6.8, whereas ANS has a negative sulfonate ion in aqueous solution[24]. The repulsion force between SF and ANS also made them difficult to be closely bound.
The assembly process was further investigated via TEM. As pH was decreased, the morphology of SF transitioned from spherical to dendritic aggregates. At pH 4.8, the aggregates formed by rod-like structures appeared (Fig. 3a and Fig. 3b), whereas at pH 6.8, heterogeneously sized micelles with diameters ranging from several tens of nanometers to hundreds of nanometers were observed (Fig. 3d). Previous studies identified micelles at neutral pH[23]. These structures might be assembled extrinsically by the aggregation of solubilized globular protein chains driven by their amino acid hydrophobicity. At pH 5.2, irregular and patchy aggregates can be seen in the image (Fig. 3c).
The results of AFM were basically consistent with those of TEM. At pH 4.8, SF proteins assembled into granules and displayed a tendency to join into bundles of fibrils (Fig. 4a). Moreover, the length of the fibrils reached micron scale. At pH 5.2 and 4.8, only particle-like aggregates can be seen, and the proportion of larger aggregates was higher at pH 5.2.
Differences among the FTIR spectra of SF under different pH values were not remarkable. As shown in Fig. S4, the peak at 1650 (amide I) and 1545 cm−1 (amide II) were associated with the random coil of SF, whereas the peak around 1630 (amide I), 1530 (amide II), and 1245 cm−1 (amide III) were characteristic of β-sheet structure. Both random coil and β-sheet structure existed in the solutions under different pH values. The peak at 1245 cm−1 was more obvious at pH 4.8 than at pH 6.8. This result may be attributed to the low β-sheet content in the aqueous solution.
CD spectra were used to verify the second structure of SF under different pH values. At pH 6.8, the CD spectra demonstrated a strong negative band centered around 193 nm and a negative one at 215 nm, indicating the dominance of random-coil structure. However, when pH decreased to 4.8, the two bands drastically inverted to a positive band at 195 nm and to a negative band at∼215 nm, indicating the emergence of β-sheet conformation. The CD spectra indicated a decrease in the content of random coil and an increase in β-sheet.