title: Primary structure elements of spider dragline silks and their contribution to protein solubility.
abstract: Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties 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 the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins 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 the synthetic 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 vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.
authors.affiliation: Department of Biotechnology, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Huemmerich
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Helsen
authors.forename: Susanne
authors.initials: S
authors.lastname: Quedzuweit
authors.forename: Jan
authors.initials: J
authors.lastname: Oschmann
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheibel
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D003001:Cloning, Molecular; D015195:Drug Design; D005349:Fibroins / Q000138:chemical synthesis* / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D012995:Solubility; D013112:Spiders / Q000737:chemistry*; D014325:Tromethamine / Q000737:chemistry
chemical_list: D002021:Buffers; D010710:Phosphates; D017680:Potassium Compounds; D014325:Tromethamine; D005349:Fibroins; C013216:potassium phosphate; D011188:Potassium