Probing the Impact of Acidification on Spider Silk Assembly Kinetics

Abstract

Spiders utilize fine adjustment of the physicochemical conditions within its silk spinning system to regulate spidroin assembly into solid silk fibers with outstanding mechanical properties. However, the exact mechanism about which this occurs remains elusive and is still hotly debated. In this study, the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. Incubating the protein-rich MA silk gland fluid at acidic pH conditions results in the formation of silk fibers that are 10-100 μm in length and ∼2 μm in diameter as judged by optical and electron microscope methods. The in vitro spider silk assembly kinetics were monitored as a function of pH with a (13)C solid-state MAS NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation as well as the percentage of β-sheet structure in the grown fibers depend on the pH. These results confirm the importance of an acidic pH gradient along the spinning duct for spider silk formation and provide a powerful spectroscopic approach to probe the kinetics of spider silk formation under various biochemical conditions.

Ion effects on the conformation and dynamics of repetitive domains of a spider silk protein: implications for solubility and β-sheet formation

“…2B). This result suggests that the polyalanine region, which contains a predominantly random coil population based on previous studies, 10,20 is not affected by the concentration of chaotropic ions.…”

Experimental Methods for Characterizing the Secondary Structure and Thermal Properties of Silk Proteins

“…[60] The advantage of this approach is that poly(Ala) in both the fluid phase and the solid fibril can be observed simultaneously (Figure 3). This allows for the monitoring of spider silk assembly kinetics as a function of biochemical conditions; in this case, the kinetics of nucleation and fiber growth were tracked as a function of pH and time.…”

Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks

“…In the gland environment, the repetitive core regions of the spidroins are predominantly unstructured (26-29) (random coil), whereas the termini exhibit pH-sensitive helical bundles (8,14,30). The pH sensitivity of the termini has been implicated as an important characteristic for the assembly of spider silk (8,14,30,31). The hydropathy plots for the spidroins are roughly sinusoidal in form, with rapidly alternating hydrophilic-hydrophobic units (approximately +0.3 to −0.6; SI Appendix, Fig.…”