用DFT和固体NMR研究丝蛋白体系中氢键、构象与功能的关系

The spinning mechanism of natural silk has been an open issue. In this study, both the conformation transition from random coil to b-sheet and b-sheet aggregation growth of silk fibroin are identified in the Bombyx mori regenerated silk fibroin aqueous solution by the circular dichroism (CD) spectroscopy. A nucleation-dependent aggregation mechanism, similar to that found in viral protein denaturation of neurodegenerative diseases, is suggested for such a structural protein system in the silk spinning process. Two steps are involved in this mechanism: (i) nucleation, a rate-limiting step involving the random coil to b-sheet transition as well as b-sheet aggregation, i.e., the formation of a nucleus or seed; (ii) aggregation growth which follows a first order kinetic process with respect to the random coil concentration. The increase of temperature accelerates the b-sheet aggregation growth if the b-sheet seed was introduced into the random coil solution. Both the silkworm and spider silk spinning processes are proposed to occur via a nucleation-dependent aggregation pathway. This work enhances our understanding of the natural silk spinning process in vivo..In addition, we investigated the effect of metal ion (Ca2+, K+, Cu2+, Zn2+) on the conformation transition of silk fibroin of Bombyx mori silkworm for viewing insight into the origin of nucleus formation. Both results from 13C-NMR and Raman spectroscopy showed that the existence of metal ions induces the transformation from coil chain to b-sheet. The reason why K+ and Ca2+ can induce b-sheet formation is that they destory the intra-chain hydrogen bonds in random coil chain, making the silk fibroin chain stretch, providing the probability of forming more regular b-sheet. But the reason why Zn2+ and Cu2+ induce b-sheet formation is that they can form new turns by foming planar four coordination complex with histidine-involved residues, making the side chains of turns form inter-chain hydrogen bond, therefore initiating b-sheet formation. The EPR results also demonstrated this possibility. The different mechanism between K+, Ca2+ and Cu2+, Zn2+ might make them exist with different contents in silk fibroin..Also in this work, a density functional theory (DFT) approach was used to assess those available structural parameters based on the comparison of calculated 13C chemical shifts and shielding tensors with experimental ones. The results indicate that: (i) silk I form, firstly be proposed in this work, is similar to a 31-helix conformation with torsion angles of <j> = -59 ± 2°, <y> = 119 ± 2°, <w> = 178 ± 2° for alanine residue and <j> = -78 ± 2°, <y> = 149 ± 2°, <w> = 178 ± 2° for glycine residue in the silk fibroin; (ii) silk II structures individually determined by Marsh, Fossey and Asakura are considered to be more rational than those determined by other authors. The resultant torsion angle ranges for silk II form are <j> = -143 ± 6°, <y> = 142 ± 5° and <w> = 178 ± 2° both for Ala and Gly residues.

本课题拟对蚕丝蛋白体系中氢键以及由此导致的构想与功能的关系进行深入的研究.利用对Ｐ偷鞍锥嗵醇八康鞍滋逑档腄FT化学位移量化计算以及分子设计与固体C-13 NMR的方法,玫角逦睦砺劢峁褂氲绾煞植纪枷?从分子水平上将结构与功能相关联,为合成性能优于蚕康男滦突撕蜕锊牧咸峁├砺壑傅?并将丝蛋白体系的共性规律推广到其他蛋白体系.

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