Pre-steady-state kinetic analysis of the elongation of amyloid fibrils of beta(2)-microglobulin with tryptophan mutagenesis.
Amyloid fibrils elongate seed dependently, with preformed fibrils providing a template for propagation of amyloidogenic conformation. Most seeding experiments use relatively few seed fibrils in comparison with monomers, resembling steady-state enzyme kinetics. Pre-steady-state kinetics should also be useful for characterizing the elongation process. With beta(2)-microglobulin (beta(2)-m), a protein responsible for dialysis-related amyloidosis, we measured the pre-steady-state kinetics of fibril elongation at pH 2.5, conditions under which the monomer is largely unfolded. beta(2)-m has Trp residues at positions 60 and 95. We used three single Trp mutants and fluorescence spectroscopy to study structural change upon fibril elongation. To focus on conformational change in monomers, we prepared seeds with a mutant without a Trp residue. At a fixed concentration of monomeric beta(2)-m, the apparent rate of fibril elongation increased with an increase in the concentration of seeds and then saturated, suggesting the accumulation of a rate-limiting intermediate. Importantly, saturation occurred at a seed/monomer ratio of around 10, as expressed by the concentration of the monomer. Because the number of monomers constituting the seed fibrils is much larger than 10, the results suggest that the elongation process is limited by "non-active-site binding." Spectral analysis indicated that, upon this non-active-site binding, both Trp60 and Trp95 are exposed to the solvent, and then only Trp60 is buried upon transition to the fibrils. We propose a new model of fibril elongation in which non-active-site binding plays a major role.
Chatani
Eri
E
Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
Ohnishi
Reina
R
Konuma
Tsuyoshi
T
Sakurai
Kazumasa
K
Naiki
Hironobu
H
Goto
Yuji
Y
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry*; D005453:Fluorescence; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D012492:Salts / Q000737:chemistry; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000235:genetics
D000682:Amyloid; D012492:Salts; D001613:beta 2-Microglobulin