Aggregation of a slow-folding mutant of a beta-clam protein proceeds through a monomeric nucleus.

Mechanistic understanding of protein aggregation, leading either to structured amyloid fibrils or to amorphous inclusion body-like deposits, should facilitate the identification of potential therapeutic intervention strategies for the devastating amyloid-based diseases. Here we focus on the in vitro aggregation of a slow-folding mutant of the beta-clam protein, cellular retinoic acid-binding protein I (P39A CRABP I), which forms inclusion bodies when expressed in Escherichia coli. Aggregation was monitored by observing the fluorescence of a fluorescein-based biarsenical dye (FlAsH) that ligates to a tetra-Cys motif, here incorporated into a flexible Omega-loop. The fluorescence signal of FlAsH on the tetra-Cys-containing P39A CRABP I is sensitive to whether this protein is native or unfolded, and was used in combination with other techniques to follow aggregate formation. The aggregation time course is compatible with a nucleation-dependent polymerization model, and detailed kinetic analysis showed that the energetically unfavorable nucleus is monomeric. A similar conclusion was reached previously for poly(Gln) species [Chen, S., Ferrone, F. A., and Wetzel, R. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889] and points to an unfavorable equilibrium between the misfolded intermediate and the bulk pool of monomers as causative in aggregation. The P39A mutation, which removes a helix-stop signal, may slow closure of the beta-barrel in P39A CRABP I relative to the wild type, leaving it vulnerable to aggregation. Wide-angle X-ray scattering showed that the amorphous aggregates formed by the aggregation-prone intermediates of P39A CRABP I contain predominantly beta-strands structured in a lamellar fashion with 10.03 A spacing between adjacent beta-sheets.