title: Fluorescence Correlation Spectroscopy: A Tool to Study Protein Oligomerization and Aggregation In Vitro and In Vivo.
abstract: Fluorescence correlation spectroscopy (FCS) is a highly sensitive analytical technique used to measure dynamic molecular parameters, such as diffusion time (from which particle size can be calculated), conformation, and concentration of fluorescent molecules. It has been particularly powerful in characterizing size distributions in molecular associations (e.g., dimer/multimer formation) both in well-behaved thermodynamically equilibrated systems in vitro as well as in more complex environments in vivo. Protein aggregation reactions like amyloid formation, in contrast, are complex, often involving a series of uniquely structured aggregation intermediates appearing at different time scales. Nonetheless, FCS can be used in appropriate cases to characterize the early stages of some aggregation reactions. Here are described step-by-step protocols and experimental procedures for the study of molecular complex formation in aggregation systems as observed in simple buffer systems, cell extracts, and living cells. The methods described are illustrated with examples from studies of the self-assembly of huntingtin fragments, but in principle can be adapted for any aggregating system.
authors.affiliation: Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, 3501 5th Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Bankanidhi
authors.initials: B
authors.lastname: Sahoo
authors.affiliation: Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, 3501 5th Avenue, Pittsburgh, PA, 15260, USA.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Drombosky
authors.affiliation: Department of Structural Biology and Pittsburgh Institute for Neurodegenerative Diseases, University of Pittsburgh School of Medicine, Biomedical Science Tower 3, 3501 5th Avenue, Pittsburgh, PA, 15260, USA. rwetzel@pitt.edu.
authors.forename: Ronald
authors.initials: R
authors.lastname: Wetzel
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D006801:Humans; D000071058:Huntingtin Protein; D046911:Macromolecular Substances / Q000737:chemistry*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics; D066263:Protein Aggregation, Pathological / Q000235:genetics*; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D000682:Amyloid; C086055:HTT protein, human; D000071058:Huntingtin Protein; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins
keywords: Amyloid; Brightness; Diffusion; Fluorescence correlation spectroscopy; Huntington’s disease; Oligomer; Particle size; Protein aggregation