Primary Structure Elements of Spider Dragline Silks and Their Contribution to Protein Solubility

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• 作者：Daniel Huemmerich ; Christopher W. Helsen ; Susanne Quedzuweit ; Jan Oschmann ; Rainer Rudolph ; and Thomas Scheibel
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：October 26, 2004
• 年：2004
• 卷：43
• 期：42
• 页码：13604 - 13612
• 全文大小：461K
• 年卷期：v.43,no.42(October 26, 2004)
• ISSN：1520-4995

文摘

Spider silk proteins have mainly been investigated with regard to their contribution to mechanicalproperties of the silk thread. However, little is known about the molecular mechanisms of silk assembly.As a first step toward characterizing this process, we aimed to identify primary structure elements of thegarden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determineprotein solubility. In addition, we investigated the influence of conditions involved in mediating naturalthread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using acloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authenticgene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesizedproteins revealed that single primary structure elements have diverse influences on protein characteristics.Repetitive regions representing the largest part of dragline silk proteins determined the solubility of thesynthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors,such as acidification and increases in phosphate concentration, which promote silk assembly in vivogenerally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineeredproteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that theseregions might play an important role in initiating assembly of spider silk proteins.