Conformational analysis of a genetically encoded FRET biosensor by SAXS

Haydyn D.T. Mertens, Alen Piljić, Carsten Schultz, Dmitri I. Svergun

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Genetically encoded FRET (Foerster resonance energy transfer) sensors are exciting tools in modern cell biology. Changes in the conformation of a sensor lead to an altered emission ratio and provide the means to determine both temporal and spatial changes in target molecules, as well as the activity of enzymes. FRET sensors are widely used to follow phosphorylation events and to monitor the effects of elevated calcium levels. Here, we report for the first time, to our knowledge, on the analysis of the conformational changes involved in sensor function at low resolution using a combination of in vitro and in cellulo FRET measurements and small-angle scattering of x rays (SAXS). The large and dynamic structural rearrangements involved in the modification of the calcium- and phosphorylation-sensitive probe CYNEX4 are comprehensively characterized. It is demonstrated that the synergistic use of SAXS and FRET methods allows one to resolve the ambiguities arising due to the rotation of the sensor molecules and the flexibility of the probe.

Original languageEnglish (US)
Pages (from-to)2866-2875
Number of pages10
JournalBiophysical Journal
Volume102
Issue number12
DOIs
StatePublished - Jun 20 2012

ASJC Scopus subject areas

  • Biophysics

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