Sammanfattning

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. (C) 2010 Elsevier Ltd. All rights reserved.

Nyckelord

spidroins; pH dependence; electrospray ionization mass spectrometry; protein-protein interactions; hydrogen/deuterium exchange

Publicerad i

Journal of Molecular Biology
2010, Volym: 404, nummer: 2, sidor: 328-336
Utgivare: ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD

SLU författare

• Askarieh, Glareh

  • Institutionen för molekylärbiologi, Sveriges lantbruksuniversitet
    
  • Universitetet i Oslo (UiO)

• Nordling, Kerstin

  • Institutionen för husdjurens biovetenskaper, Sveriges lantbruksuniversitet
    

• Hedhammar, My

  • Institutionen för husdjurens biovetenskaper, Sveriges lantbruksuniversitet
    

• Rising, Anna

  • Institutionen för husdjurens biovetenskaper, Sveriges lantbruksuniversitet
    

• Knight, Stefan David

  • Institutionen för molekylärbiologi, Sveriges lantbruksuniversitet
    

• Johansson, Jan

  • Institutionen för husdjurens biovetenskaper, Sveriges lantbruksuniversitet
    

UKÄ forskningsämne

Biokemi och molekylärbiologi


Publikationens identifierare

DOI: https://doi.org/10.1016/j.jmb.2010.09.054

Permanent länk till denna sida (URI)

https://res.slu.se/id/publ/60992