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Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation
(Karolinska Institutet, Department of Neurobiology)
(Latvian Institute of Organic Synthesis, Riga, Latvia)
KTH, School of Engineering Sciences (SCI), Applied Physics, Experimental Biomolecular Physics. (Experimentell biomolekylär fysik)
(Karolinska Institutet, Department of Neurobiology, Stockholm, Sweden)
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2014 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, 3254- p.Article in journal (Refereed) Published
Abstract [en]

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

Place, publisher, year, edition, pages
Nature Publishing Group, 2014. Vol. 5, 3254- p.
Keyword [en]
animal; biosynthesis; chemistry; dimerization; genetics; metabolism; nuclear magnetic resonance spectroscopy; pH; spectrofluorometry; spider; static electricity
National Category
Biochemistry and Molecular Biology
Research subject
Biological Physics
Identifiers
URN: urn:nbn:se:kth:diva-187706DOI: 10.1038/ncomms4254ISI: 000332667600001OAI: oai:DiVA.org:kth-187706DiVA: diva2:931211
Funder
Danish National Research FoundationSwedish Research Council
Note

QC 20160527

Available from: 2016-05-26 Created: 2016-05-26 Last updated: 2017-11-30Bibliographically approved
In thesis
1. Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
Open this publication in new window or tab >>Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence spectroscopy and imaging have a very broad spectrum of applicationswithin the life sciences, in particular for detection and characterization ofbiomolecular dynamics and interactions in different environments. This thesis comprisesprojects that strive to further expand the information content extracted fromthe detected fluorescence, leading to sensitive readout parameters for studies ofbiomolecular dynamics and interactions. Two major strategies are presented toachieve this aim. The first strategy is based on the expansion of the availablereadout parameters beyond the "traditional" fluorescence parameters: intensity,wavelength, polarization and fluorescence lifetime. The additional parameters arebased on blinking properties of fluorescent labels. In particular on transitions betweensinglet and triplet states, and transitions between the trans- and cis-isomersof fluorophores. Two publications in the thesis are based on this strategy (paperI and IV). The second strategy is based on the utilization of fluorescence intensityfluctuations in order to detect the oligomerization mechanisms of fluorescentlylabeled peptides and proteins. This strategy combines the intensity fluctuationanalysis and the readout of distance dependent energy transfer between fluorescentmolecules together with the correlation analysis of fluorescence from two labeledproteins emitting at different wavelengths. Another two publications presented inthe thesis are based on the second comprehensive strategy (papers II and III).The work presented in this thesis shows that the blinking kinetics of fluorescentlabels contain significant information that can be exploited by a combination of fluctuationsanalysis with distance dependent excitation energy transfer between thefluorescent molecules, or by analysis of fluorescence covariance between moleculesthat emit at different wavelengths. These fluorescence-based methods have a significantpotential for molecular interaction studies in the biomedical field.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2016. x, 59 p.
Series
TRITA-FYS, ISSN 0280-316X ; 2016:22
Keyword
FCS, FCCS, Isomerization, TRAST, NMR, FRET, biomombrane, fluidity
National Category
Biophysics
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-187708 (URN)978-91-7729-026-1 (ISBN)
Public defence
2016-06-13, FB52, KTH, AlbaNova University Center, Roslagsvägen 30 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20160527

Available from: 2016-05-27 Created: 2016-05-26 Last updated: 2016-05-31Bibliographically approved

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Wennmalm, StefanWidengren, Jerker

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