title: Assessing mutant huntingtin fragment and polyglutamine aggregation by atomic force microscopy.
abstract: Huntington disease (HD), a neurodegenerative disorder, is caused by an expansion of more than 35-40 polyglutamine (polyQ) repeats located near the N-terminus of the huntingtin (htt) protein. The expansion of the polyQ domain results in the ordered assembly of htt fragments into fibrillar aggregates that are the main constituents of inclusion bodies, which are a hallmark of the disease. This paper describes protocols for studying the aggregation of mutant htt fragments and synthetic polyQ peptides with atomic force microscopy (AFM). Ex situ AFM is used to characterize aggregate formation in protein incubation as a function of time. Methods to quickly and unambiguously distinguish specific aggregate species from complex, heterogeneous aggregation reactions based on simple morphological features are presented. Finally, the application of time lapse atomic force microscopy in solution is presented for studying synthetic model polyQ peptides, which allows for tracking the formation and fate of individual aggregates on surfaces over time. This ability allows for dynamic studies of the aggregation process and direct observation of the interplay between different types of aggregates.
authors.affiliation: The C. Eugene Bennett Department of Chemistry, West Virginia University, 217 Clark Hall, P.O. Box 6045, Morgantown, WV 26506, USA.
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: Burke
authors.forename: Jordan
authors.initials: J
authors.lastname: Godbey
authors.forename: Justin
authors.initials: J
authors.lastname: Legleiter
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000473:pathology; D007091:Image Processing, Computer-Assisted; D018625:Microscopy, Atomic Force; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification; D009687:Nuclear Proteins / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D059008:Time-Lapse Imaging
chemical_list: C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins