A pH-dependent dimer lock in spider silk protein

• Spider dragline silk, one of the strongest polymers in nature, is composed of proteins termed major ampullate spidroin (MaSp) 1 and MaSp2. The N-terminal (NT) domain of MaSp1 produced by the nursery web spider Euprosthenops australis acts as a pH-sensitive relay, mediating spidroin assembly at around pH 6.3. Using amide hydrogen/deuterium exchange combined with mass spectrometry (MS), we detected pH-dependent changes in deuterium incorporation into the core of the NT domain, indicating global structural stabilization at low pH. The stabilizing effects were diminished or abolished at high ionic strength, or when the surface-exposed residues Asp40 and Glu84 had been exchanged with the corresponding amides. Nondenaturing electrospray ionization MS revealed the presence of dimers in the gas phase at pH values below--but not above--6.4, indicating a tight electrostatic association that is dependent on Asp40 and Glu84 at low pH. Results from analytical ultracentrifugation support these findings. Together, the data suggest a mechanism whereby lowering the pH to <6.4 results in structural changes and alteration of charge-mediated interactions between subunits, thereby locking the spidroin NT dimer into a tight entity important for aggregation and silk formation.

Ämnesord

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Amino Acid Sequence

Amino Acid Substitution

Animals

Deuterium

Dimerization

Fibroins/*chemistry/genetics

Hydrogen-Ion Concentration

Models

Molecular

Molecular Sequence Data

Mutagenesis

Site-Directed

Osmolar Concentration

Protein Multimerization

Protein Stability

Protein Structure

Quaternary

Protein Structure

Tertiary

Protein Subunits

Recombinant Proteins/chemistry/genetics

Spectrometry

Mass

Electrospray Ionization

Spiders/chemistry/genetics

Static Electricity

Ultracentrifugation

Publikations- och innehållstyp

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