Interaction Of Bombyx Mori Silk Fibroin With Mn(Ⅱ) Studied Using Magnetic Resonance Methods And Biosynthesis Of Amyloid-β 42 Peptide

More and more applications of natural silk as a biomedical material,which is produced by a wide variety of insects like silkworms and spiders,have attracted our attention mostly.The formation of natural silk fiber depends on the conformational transition of the silk fibroin(SF) from a soluble random coil and/or helix state(Silkâ… ) to an insolubleβ-sheet state(Silkâ…¡).Previous investigations have demonstrated that K⁺,Na⁺,Ca²⁺,Cu²⁺ ions at the appropriate concentrations could facilitate the transition.Mn(â…¡) ion is one of the metal ions existing in silkworm gland and silk fiber.However,the role of Mn(â…¡) in the gland and silk fiber and the coordination of Mn(â…¡) with silk fibroin are unclear.Therefore,we used nuclear magnetic resonance (NMR) and electron paramagnetic resonance(EPR) spectroscopy to study the role of Mn(â…¡) in silk fibroin.In the present investigation we report that Mn²⁺ ion has no detectable overall effect on the conformational transition,as demonstrated by ¹³C NMR spectroscopy.As suggested by EPR study,Mn(â…¡) ions are likely to exist in the silk fibroin molecules in two ways:a six-coordination Mn(â…¡)/SF complex and a Mn(H₂O)₆²⁺ complex.Mn(â…¡) binding the amino acid residues which locate in the hydrophilic spacers of the silk fibroin heavy chain might allow the silk fibroin to favor the conformation in Silkâ…¡state while the Mn(H₂O)₆²⁺ complex might have an ability to stabilize the silk fibroin in Silkâ… state in some extent.EPR spectra of Bombyx mori silk fibroin(SF) were measured,and showed a singlet peak at g = 2.0030±0.0005,which was thought to be a signal of the natural free radical in the heavy chain of the silk fibroin.Theoretical ¹H hyperfine coupling constants calculated by density functional theory(DFT) and theoretical EPR spectrum calculated by EasySpin indicated that the free radicals in SF were the tyrosyl radicals and they likely resided in the domain of Silkâ… conformation.In addition,the calculation demonstrated that the EPR spectrum of tyrosyl radicals was dependent sensitively on the local structure of protein.The EPR signal of tyrosyl radical is isotropic in the Silkâ… conformation,while it is anisotropic in Silkâ…¡conformation, which suggests that the tyrosyl radical could be used as an appropriate probe to detect the structural information of the proteins.Using ¹³C solid-state NMR we studied the structures of two spidroin-like copolymers that were synthesized by the copolymerization of polyalanine((Ala)₅) with oligomers of polystyrene(PS,M_w=2000) and polyisoprene(PI,Mw=2210).¹³C CP/MAS NMR spectra and spin-lattice relaxation time(T_1ρ(¹³C)) results of the copolymers indicate that the chemical shifts of(Ala)₅ in both copolymers of polystyrene-co-polyalanine(PS-co-PAL) and polyisoprene-co-polyalanine (PI-co-PAL) were almost the same.This means that the(Ala)₅ in the two copolymers has similar chemical environments and secondary structures.The similar T_1ρ(¹³C) values for(Ala)₅ in the two copolymers indicates that(Ala)₅ peptide segments also have similar aggregate structures.However,the mechanical properties of the two spidroin-like copolymers are quite different.PS-co-PAL is granular and tough while PI-co-PAL is rubber-like and tensible at room temperature.This indicates that performances of spidroin-like copolymers are strongly linked to the properties of the chosen polymers.In addition,we biosynthesized human amyloid-β42(Aβ42) peptide. Tricine-SDS-PAGE and MOLDI-TOF/MS/MS results proved the peptide we obtained to be the human Aβ42.Circular dichroism result proved that Aβ42 preserved the conformation of random coil mainly.