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Verfasst von:Milles, Sigrid [VerfasserIn]   i
 Tyagi, Swati [VerfasserIn]   i
 Banterle, Niccolò [VerfasserIn]   i
 Köhler, Christine [VerfasserIn]   i
 Plass, Tilman [VerfasserIn]   i
 Lemke, Edward A. [VerfasserIn]   i
Titel:Click strategies for single-molecule protein fluorescence
Verf.angabe:Sigrid Milles, Swati Tyagi, Niccolò Banterle, Christine Koehler, Virginia VanDelinder, Tilman Plass, Adrian P. Neal, and Edward A. Lemke
E-Jahr:2012
Jahr:February 22, 2012
Umfang:9 S.
Fussnoten:Gesehen am 20.09.2018
Titel Quelle:Enthalten in: American Chemical SocietyJournal of the American Chemical Society
Ort Quelle:Washington, DC : ACS Publications, 1879
Jahr Quelle:2012
Band/Heft Quelle:134(2012), 11, Seite 5187-5195
ISSN Quelle:1520-5126
Abstract:Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.
DOI:doi:10.1021/ja210587q
URL:Bitte beachten Sie: Dies ist ein Bibliographieeintrag. Ein Volltextzugriff für Mitglieder der Universität besteht hier nur, falls für die entsprechende Zeitschrift/den entsprechenden Sammelband ein Abonnement besteht oder es sich um einen OpenAccess-Titel handelt.

Volltext ; Verlag: http://dx.doi.org/10.1021/ja210587q
 Volltext: https://doi.org/10.1021/ja210587q
 DOI: https://doi.org/10.1021/ja210587q
Datenträger:Online-Ressource
Sprache:eng
K10plus-PPN:1581164653
Verknüpfungen:→ Zeitschrift

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