Research On Preparation And Assembly Behavior Of Silk Peptides

Self-assembly of peptides is now emerging as an important and fast developing field. By self-assembly, the molecule chains of peptide combine with each other to form promising nanomaterials with specific structures and functions. These nanomaterials, including nanofiber, hydrogel, nanotube, micelle, surfactant and so on, can be applied in various fields, such as medicine, tissue engineering, drug release, and biological surface engineering, etc.Generally, the peptide building blocks for self-assembly are mainly obtained by chemical synthesis or genetic engineering. With a typical ’bottom-up’ way, researchers can design the peptides at the molecular level, and control their formation and configuration. However, such technologies are costly and time consuming which prevents the applications of peptides, especially those used for self-assembly. Thus, it is meaningful to find a fast and cost-effective alternative way to obtain scale-up peptides.In the present study, we used a novel ’top-down’ method to obtain peptides for self-assembly. That is, get large-scale products of short peptides from silk fibroin by enzymatic hydrolysis. The Bombyx mori silkworm silk was chosen as the raw materials, due to its rich resource, low cost and high repetitive motifs in the protein. Silk fibroin was hydrolyzed by several kinds of enzymes, and the produced silk peptides were purified by RP-HPLC. Finally, the assembly behavior of the produced silk peptides was investigated.To obtain peptides for self-assembly, a-Chymotrpsin and Elastine were chosen to hydrolyze the silk fibroin in aqueous solution respectively. a-Chymotrpsin cleaves the carboxyl sites of Phe, Tyr and Trp, these amino acids mainly locating in non- crystalline section of fibroin, while Elastine degrades the carboxyl sites of Gly and Ala, these amino acids locating both in non-crystalline section and crystalline section of fibroin. The produced silk peptides by hydrolysis were collected and purified by RP-HPLC, and then characterized with ESI-MS, MALDI-TOF-MS, NMR and protein sequencing.The assembly behavior of one produced silk peptides (SFC-1, GAGSGAGAGSGA) from crystalline section was investigated. The results indicated that this peptide could form nanofibers in aqueous solution and displayed a gel-like feature at high concentration. The morphology of the nanofiber was investigated by AFM, SEM, TEM and cyro-SEM, and the secondary structure was characterized by FTIR, CD, fluorescence and XRD. The results verified the cross-β feature of SFC-1. Meanwhile, we evaluated the effect of temperature on the secondary structure of SFC-1, and found that it could recover its secondary structure after heat-denaturing. The formation of nanofibers required the participation of β-sheet structure, besides affection by the length of peptide, the amino acid composition, etc. SFC-1was also used as a template to reduce gold. The gold nanoparticles with3nm in diameter could align along the nanofibers, which indicated the potential of the peptide in synthesizing one dimensional inorganic nanomaterials.Interestingly, another peptide (SFA-1, GAGAGAGY) from the non-crystalline section showed a totally different assembling behavior compared to SFC-1. FTIR and CD spectra indicated it mainly adopted random coil when its aqueous solution was directly dried on a solid surface. This could also be verified by the random particles in the AFM images. The peptide could assemble into nanofibers with its second structure dominated by β-sheet, after the treatment with n-butanol-H2O solution (n-butanol:H2O=9:1(v/v)) and dry on a solid surface. The n-butanol-H2O solution could promote SFA-1molecular to arrange regularly and form nanofibers on solid surface. A similar result was also observed when formic acid was used as solvent.Finally, the effect of variation of single amino acid on the assembly was investigated. The assembly behavior of a peptide GAGAGAGS (obtained through solid phase synthesis) was compared with SFA-1. Unlike SFA-1which retained random coil in aqueous solution all the time, a transition from random coil to β-sheet was observed for GAGAGAGS in aqueous solution by CD. The corresponding AFM and SEM images showed the change of GAGAGAGS from particle-like aggregates to nanofibers, and the entwine behavior between fibers was also observed. All the results suggested that the differences of the assembly for two peptides arise from the steric effect of the amino acid at C-terminal:Tyr and Ser, and the aromatic ring in tyrosine might hinder the fibrillization of the peptide.