Beating the heat--fast scanning melts silk beta sheet crystals

Abstract

Beta-pleated-sheet crystals are among the most stable of protein secondary structures, and are responsible for the remarkable physical properties of many fibrous proteins, such as silk, or proteins forming plaques as in Alzheimer's disease. Previous thinking, and the accepted paradigm, was that beta-pleated-sheet crystals in the dry solid state were so stable they would not melt upon input of heat energy alone. Here we overturn that assumption and demonstrate that beta-pleated-sheet crystals melt directly from the solid state to become random coils, helices, and turns. We use fast scanning chip calorimetry at 2,000 K/s and report the first reversible thermal melting of protein beta-pleated-sheet crystals, exemplified by silk fibroin. The similarity between thermal melting behavior of lamellar crystals of synthetic polymers and beta-pleated-sheet crystals is confirmed. Significance for controlling beta-pleated-sheet content during thermal processing of biomaterials, as well as towards disease therapies, is envisioned based on these new findings.

Figure 1. Silk Fibroin Structure Before and After Assumed Thermal Melting.

(a) A three-chain silk sequence is depicted based on the amino acid hexamer, GAGAGS, with anti-parallel molecular chain axes. The chain comprises atoms of carbon (gray), nitrogen (blue), oxygen (red), and hydrogen (white), which have intermolecular hydrogen bonding (represented as three short vertical lines). (b) The molecular chains stack together to form the three-dimensional, beta pleated sheet crystal. This secondary structure is formed by spiders and silkworms naturally during fiber spinning from the solution state, which process converts random coils, turns, and helices into three-dimensional crystals. (c) The temperature vs. time profile is shown during fast thermal treatment, which causes the beta pleated sheet crystals (at point A) to become melted within a few tenths of a second, reversing the natural fiber spinning process, and restoring the material to the non-crystalline solid state (at point B). The on-chip melting uses heating and cooling rates of thousands of degrees per second.

Figure 2. Comparison of Thermal Characteristics of Silk and Synthetic Polymer at Different Heating Rates.

Observable transitions during first (1, red) and second (2, blue) heating include glass transition (T_g), crystallization exotherm (T_c), melting endotherm (T_m), and thermal degradation (T_d). Endotherms are presented with downward deflection. (a) Film of reconstituted B. mori silk fibroin protein during DSC heating at 2 K/min. The sample degrades and loses mass upon first heating. The second heating scan does not retrace the first, and no glass transition or crystallization can be observed. (b) Synthetic polymer (exemplified by isotactic polystyrene, iPS) during DSC heating at 2 K/min. Sample does not degrade after heating to 538 K, and the overlapping second heating trace shows the same thermal transitions. (c) Film of reconstituted B. mori silk fibroin protein, now heated at 2,000 K/s using fast scanning chip calorimetry, shows melting of beta pleated sheet crystals. The second trace, after cooling from the melt at 2,000 K/s, confirms the non-crystalline nature of the film after beta-pleated-sheets were melted during the first scan. (d, e) Sample from (c), imaged at room temperature with unpolarized light using a 20× objective on the flat, optically transparent sensor, before (d) and after (e) fast heating to 650 K. As a result of melting the film changes shape slightly.

Figure 3. Comparison of Structural and Fast Scanning Calorimetric Data.

Room temperature FTIR absorbance spectra of silk fibroin (a) with corresponding calorimetric data at 2000 K/s (b). (a) Initial non-crystalline silk (M0). Silk exposed in-situ three times to MeOH (C1–C3 red: 18 h – C1; 2.5 h – C2; 20 min – C3) crystallizes, and before fast heating contains beta-pleated-sheet crystals, with characteristic beta sheet absorbance marked at 1629 cm⁻¹. After fast heating to 650 K, beta-pleated-sheet crystals melt and only random coils, turns, and alpha helices remain (M1–M3 blue). (b) Heat capacity vs. temperature at 2000 K/s for the same film shown in (a), presented as matched sets. Initial non-crystalline silk (M0). MeOH-exposed crystalline samples (C1–C3 red) during first heating. After cooling, the film was immediately reheated (M1–M3 blue). Absence of melting endotherms in reheating scans confirms silk is no longer crystalline. (c) Heat capacity vs. temperature at 2000 K/s, for water annealed silk fibroin film and a bundle of native degummed cocoon fibers. Down arrow marks T_g; up arrow marks onset of melting; horizontal black arrow marks region of fiber melting and shape change to droplet morphology. (d, e) A bundle of degummed cocoon fibers, imaged in unpolarized light at 20× magnification before (d) and after (e) heating to 690 K. The separate fibers contract when the beta-pleated-sheet crystals melt, removing the physical crosslinks, and allowing the fibers to coalescence into a single droplet.

Figure 4. Silk Structure Before and After Melting.

A single degummed native cocoon fiber before (a) and after (b) melting at 2000 K/s, imaged between the polarizer (vertical) and the analyzer (80 degrees to the polarizer) at 10× magnification. Suggested secondary structure is indicated in the lower panels: beta-pleated-sheet crystals in degummed native cocoon fiber (c) and random coils, turns and alpha helices after melting (d). The figure demonstrates the main results of this work, that silk can be melted directly from the solid state upon the input of heat energy alone, without degradation.