Pre-Steady-State Kinetic Analysis of the Elongation of Amyloid Fibrils of β2-Microglobulin with Tryptophan Mutagenesis

摘要: Abstract Amyloid fibrils elongate seed dependently, with preformed providing a template for propagation of amyloidogenic conformation. Most seeding experiments use relatively few in comparison monomers, resembling steady-state enzyme kinetics. Pre-steady-state kinetics should also be useful characterizing the elongation process. With β 2 -microglobulin (β -m), protein responsible dialysis-related amyloidosis, we measured pre-steady-state fibril at pH 2.5, conditions under which monomer is largely unfolded. -m has Trp residues positions 60 and 95. We used three single mutants fluorescence spectroscopy to study structural change upon elongation. To focus on conformational prepared seeds mutant without residue. At fixed concentration monomeric -m, apparent rate increased an increase then saturated, suggesting accumulation rate-limiting intermediate. Importantly, saturation occurred seed/monomer ratio around 10, as expressed by monomer. Because number monomers constituting much larger than results suggest that process limited “non-active-site binding.” Spectral analysis indicated that, this non-active-site binding, both Trp60 Trp95 are exposed solvent, only buried transition fibrils. propose new model binding plays major role.