TY - JOUR
T1 - A pH-Dependent Dimer Lock in Spider Silk Protein
AU - Landreh, Michael
AU - Askarieh, Glareh
AU - Nordling, Kerstin
AU - Hedhammar, My
AU - Rising, Anna
AU - Casals, Cristina
AU - Astorga-Wells, Juan
AU - Alvelius, Gunvor
AU - Knight, Stefan David
AU - Johansson, Jan
AU - Jörnvall, Hans
AU - Bergman, Tomas
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - spidroins
KW - pH dependence
KW - electrospray ionization mass spectrometry
KW - protein-protein interactions
KW - hydrogen/deuterium exchange
KW - spidroins
KW - pH dependence
KW - electrospray ionization mass spectrometry
KW - protein-protein interactions
KW - hydrogen/deuterium exchange
UR - https://res.slu.se/id/publ/60992
U2 - 10.1016/j.jmb.2010.09.054
DO - 10.1016/j.jmb.2010.09.054
M3 - Journal article
C2 - 20887730
SN - 0022-2836
VL - 404
SP - 328
EP - 336
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -