Poly-(L-alanine) expansions form core beta-sheets that nucleate amyloid assembly.
Expansion to a total of 11-17 sequential alanine residues from the normal number of 10 in the polyadenine-binding protein nuclear-1 (PABPN1) results in formation of intranuclear, fibrillar inclusions in skeletal muscle and hypothalamic neurons in adult-onset, dominantly inherited oculopharyngeal muscular dystrophy (OPMD). To understand the role that homopolymeric length may play in the protein misfolding that leads to the inclusions, we analyzed the self-assembly of synthetic poly-(L-alanine) peptides having 3-20 residues. We found that the conformational transition and structure of polyalanine (polyAla) assemblies in solution are not only length-dependent but also are determined by concentration, temperature, and incubation time. No beta-sheet complex was detected for those peptides characterized by n < 8, where n is number of alanine residues. A second group of peptides with 7 < n < 15 showed varying levels of complex formation, while for those peptides having n > 15, the interconversion process from the monomeric to the beta-sheet complex was complete under any of the tested experimental conditions. Unlike the typical tinctorial properties of amyloid fibrils, polyalanine fibrils did not show fluorescence with thioflavin T or apple-green birefringence with Congo red; however, like amyloid, X-ray diffraction showed that the peptide chains in these fibrils were oriented normal to the fibril axis (i.e., in the cross-beta arrangement). Neighboring beta-sheets are quarter-staggered in the hydrogen-bonding direction such that the alanine side-chains were closely packed in the intersheet space. Strong van der Waals contacts between side-chains in this arrangement likely account for the high stability of the macromolecular fibrillar complex in solution over a wide range of temperature (5-85 degrees C), and pH (2-10.5), and its resistance to denaturant (< 8 M urea) and to proteases (protease K, trypsin). We postulate that a similar stabilization of an expanded polyalanine stretch could form a core beta-sheet structure that mediates the intermolecular association of mutant proteins into fibrillar inclusions in human pathologies.
Shinchuk
Leonid M
LM
Department of Biology, Boston College, Chestnut Hill, Massachusetts 02467-3811, USA.
Sharma
Deepak
D
Blondelle
Sylvie E
SE
Reixach
Natalia
N
Inouye
Hideyo
H
Kirschner
Daniel A
DA
D000682:Amyloid / Q000737:chemistry*; D003224:Congo Red; D002479:Inclusion Bodies / Q000737:chemistry / Q000648:ultrastructure; D039141:Muscular Dystrophy, Oculopharyngeal / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000737:chemistry*; D011108:Polymers; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature; D014961:X-Ray Diffraction
D000682:Amyloid; D010455:Peptides; D011108:Polymers; C019529:polyalanine; D003224:Congo Red