title: Detection and analysis of protein aggregation with confocal single molecule fluorescence spectroscopy.
abstract: The misfolding and aggregation of proteins is a common phenomenon both in the cell, in in vitro protein refolding, and the corresponding biotechnological applications. Most importantly, it is involved in a wide range of diseases, including some of the most prevalent neurodegenerative disorders. However, the range of methods available to analyze this highly heterogeneous process and the resulting aggregate structures has been very limited. Here we present an approach that uses confocal single molecule detection of FRET-labeled samples employing four detection channels to obtain information about diffusivity, anisotropy, fluorescence lifetimes and Förster transfer efficiencies from a single measurement. By combining these observables, this method allows the separation of subpopulations of folded and misfolded proteins in solution with high sensitivity and a differentiation of aggregates generated under different conditions. We demonstrate the versatility of the method with experiments on rhodanese, an aggregation-prone two-domain protein.
authors: Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, Zurich, Switzerland.
authors: Frank
authors: F
authors: Hillger
authors: Daniel
authors: D
authors: Nettels
authors: Simone
authors: S
authors: Dorsch
authors: Benjamin
authors: B
authors: Schuler
mesh_terms: D000818:Animals; D002417:Cattle; D005454:Fluorescence Polarization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D018613:Microscopy, Confocal; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D011506:Proteins; D013884:Thiosulfate Sulfurtransferase