Multi-Parameter Fluorescence Spectroscopy and Imaging for Quantitative FRET Measurements

Abstract

Fluorescence spectroscopy and imaging are important biophysical techniques to study dynamics and function of biomolecules in vitro and in live cells. However, often our view of molecular function is still formed, to a significant extent, by traditional structure determination showing many detailed static snapshots of biomolecular structures. Recent fluorescence experiments added a dynamic perspective by showing the heterogeneity and flexibility of molecular structures, visualizing transiently populated conformational states and identifying exchange pathways. This dynamic personality is an essential aspect for understanding their function. Following the ideas of Theodor Forster and Gregorio Weber, I introduce multi-parameter fluorescence detection (MFD) [1] and multi-parameter fluorescence image spectroscopy (MFIS) [2]. These techniques register all eight characteristic fluorescence parameters in a single measurement so that we can gain maximum resolution of specific fluorescence information on the biomolecule. The five intrinsic fluorescence properties of a fluorophore such as spectral properties in absorption and in fluorescence, brightness and quantum yield, lifetime and anisotropy report on its local environment. Three fluorescence parameters are connected to the system. The application of fluctuation spectroscopy allows us to resolve system properties such as diffusional properties and kinetic networks. The use of more than one fluorophore per molecule opens additional opportunities arising from photon densities, coincidences and dipolar coupling by Forster Resonance Energy Transfer (FRET) to study the stoichiometry and structure of biomolecular systems. Taking the large GTPase human Guanylate binding protein 1 (hGBP1) as example, I will present the structural and dynamical characterization of all intermediates during hGBP1 oligomerization by high-precision FRET in vitro and in live cells [4]. [1] Widengren et al.; Anal. Chem. 78, 2039-2050 (2006). [2] Weidtkamp-Peters et al.; Photochem. Photobiol. Sci., 8, 470-480 (2009). [3] Kalinin et al.; Nat. Methods 9, 1218-1225 (2012). [4] Kravets et. al.; eLife 5, e11479 (2016).