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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, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik. (Experimentell biomolekylär fysik)
(Karolinska Institutet, Department of Neurobiology, Stockholm, Sweden)
Visa övriga samt affilieringar
2014 (Engelska)Ingår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, s. 3254-Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Nature Publishing Group, 2014. Vol. 5, s. 3254-
Nyckelord [en]
animal; biosynthesis; chemistry; dimerization; genetics; metabolism; nuclear magnetic resonance spectroscopy; pH; spectrofluorometry; spider; static electricity
Nationell ämneskategori
Biokemi och molekylärbiologi
Forskningsämne
Biologisk fysik
Identifikatorer
URN: urn:nbn:se:kth:diva-187706DOI: 10.1038/ncomms4254ISI: 000332667600001OAI: oai:DiVA.org:kth-187706DiVA, id: diva2:931211
Forskningsfinansiär
Danmarks GrundforskningsfondVetenskapsrådet
Anmärkning

QC 20160527

Tillgänglig från: 2016-05-26 Skapad: 2016-05-26 Senast uppdaterad: 2017-11-30Bibliografiskt granskad
Ingår i avhandling
1. Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
Öppna denna publikation i ny flik eller fönster >>Fluorescence fluctuation studies of biomolecular interactions in solutions, biomembranes and live cells
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Stockholm: KTH Royal Institute of Technology, 2016. s. x, 59
Serie
TRITA-FYS, ISSN 0280-316X ; 2016:22
Nyckelord
FCS, FCCS, Isomerization, TRAST, NMR, FRET, biomombrane, fluidity
Nationell ämneskategori
Biofysik
Forskningsämne
Biologisk fysik
Identifikatorer
urn:nbn:se:kth:diva-187708 (URN)978-91-7729-026-1 (ISBN)
Disputation
2016-06-13, FB52, KTH, AlbaNova University Center, Roslagsvägen 30 B, Stockholm, 13:00 (Engelska)
Opponent
Handledare
Anmärkning

QC 20160527

Tillgänglig från: 2016-05-27 Skapad: 2016-05-26 Senast uppdaterad: 2016-05-31Bibliografiskt granskad

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Chmyrov, VolodymyrWennmalm, StefanWidengren, Jerker
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