Alanine scanning mutagenesis of Abeta(1-40) amyloid fibril stability.
We describe here an alanine scanning mutational analysis of the Abeta(1-40) amyloid fibril monitored by fibril elongation thermodynamics derived from critical concentration values for fibril growth. Alanine replacement of most residues in the amyloid core region, residues 15-36, leads to destabilization of the elongation step, compared to wild-type, by about 1kcal/mol, consistent with a major role for hydrophobic packing in Abeta(1-40) fibril assembly. Where comparisons are possible, the destabilizing effects of Ala replacements are generally in very good agreement with the effects of Ala replacements of the same amino acid residues in an element of parallel beta-sheet in the small, globular protein Gbeta1. We utilize these Ala-WT DeltaDeltaG values to filter previously described Pro-WT DeltaDeltaG values, creating Pro-Ala DeltaDeltaG values that specifically assess the sensitivity of a sequence position, in the structural context of the Abeta fibril, to replacement by proline. The results provide a conservative view of the energetics of Abeta(1-40) fibril structure, indicating that positions 18-21, 25-26, and 32-33 within amyloid structure are particularly sensitive to the main-chain disrupting effects of Pro replacements. In contrast, residues 14-17, 22, 24, 27-31, and 34-39 are relatively insensitive to Pro replacements; most N-terminal residues were not tested. The results are discussed in terms of amyloid fibril structure and folding energetics, in particular focusing on how the data compare to those from other structural studies of Abeta(1-40) amyloid fibrils grown in phosphate-buffered saline at 37 degrees C under unstirred ("quiescent") conditions.
Graduate School of Medicine, R221, University of Tennessee, 1924 Alcoa Highway, Knoxville, TN 37920, USA.
Angela D
AD
Williams
Shankaramma
S
Shivaprasad
Ronald
R
Wetzel
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics
D000682:Amyloid; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); D000409:Alanine