Amyloid Protein Aggregation Studied at Different Resolutions: NMR, FRET, AFM, STED, Quantum Dots, Nanowells, Cells

M. JULIA ROBERTI, M. SOLEDAD CELEJ, SHYAMALA THIRUNAVUKKUARASU, CARLOS W. BERTONCINI, REINHARD KLEMENT, AVISHAY PELAH, ALEXANDER P. DEMCHENKO, ELIZABETH A. JARES-ERIJMAN

Budapest, Hungary

Workshop; Intrinsically Unfolded Proteins (IUP) Workshop 2007: Biophysical Characterisation and Biological Significance; 2007

European Molecular Biology Organization (EMBO)

A common denominator in the major neurodegenerative diseases is the pathological association of proteins adopting an aggregation-prone crossed β-sheet secondary structure, distinct from their natively unstructured or globular conformation. Parkinson's disease is characterized by formation in the midbrain of aggregates of the 140-aa protein α-synuclein (AS). Cytotoxicty is currently attributed to the action of intermediate (oligomeric) species rather than the classical end-state amyloid fibrils. A major effort in our labs is directed at elucidating features of the aggregation mechanism and devising new methods for identifying and characterizing oligomeric and fibrillar species of wildtype and familial mutant forms of AS. We utilize multiple approaches in such studies, conducted both in solution [solution and solid-state NMR, covalent and non-covalent environment-sensitive fluorescence probes, DLS, MS, electrochemistry, molecular dynamics, stopped-flow] and in microscopes [underwater temperature-controlled AFM, fluorescence (FRET, FLIM, anisotropy, hyperspectral), nanowell arrays] offering different levels of spatial and spectral resolution. Recent efforts have been based on a fusion protein of AS created for cellular studies (see ref.), and new covalent and non-covalent fluorescent probes reporting on the properties of monomeric AS, the elusive oligomers, and fibrils. We also have obtained evidence for a mixed mode of monomer addition, involving both liganded and unliganded AS species.