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PLoS Biol. 2014 Aug 5;12(8):e1001921. doi: 10.1371/journal.pbio.1001921. eCollection 2014 Aug.

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Author information

1
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
2
Institute of Biological Sciences and Biotechnology, Donghua University, Shanghai, People's Republic of China.
3
Department of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, Riga, Latvia.
4
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
5
Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Stockholm, Sweden.
6
Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.
7
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
8
Departments of Neurosurgery, Physiology and Neuroscience, New York University School of Medicine, New York, New York, United States of America.
9
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Stockholm, Sweden; Institute of Mathematics and Natural Sciences, Tallinn University, Tallinn, Estonia.
10
Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden; Department of Neurobiology, Care Sciences and Society (NVS), Karolinska Institutet, Stockholm, Sweden.

Abstract

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive β-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

PMID:
25093327
PMCID:
PMC4122339
DOI:
10.1371/journal.pbio.1001921
[Indexed for MEDLINE]
Free PMC Article
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