Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

The formation of amyloid fibrils by the SH3 domain of the a-subunit of bovi ne phosphatidylinositol-3 ' -kinase (PI3-SH3) has been investigated under c arefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low PH show that their propert ies can be correlated with the nature of the resulting aggregates defined b y EM and FTIR spectroscopy. Compact partially folded states, favoured by th e addition of anions, are prone to precipitate rapidly into amorphous speci es, whilst well-defined fibrillar structures are formed slowly from more ex panded denatured states. Kinetic data obtained by a variety of techniques s how a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solut ion during or after this lag phase. EM and FTIR indicate the presence of am orphous aggregates (protofibrils) rich in beta -structure after the lag pha se but prior to the development of well-defined amyloid fibrils. These obse rvations strongly suggest a nucleation and growth mechanism for the formati on of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the PH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the p rotofilaments forming the amyloid fibrils. Moreover, different hydrogen bon ding patterns related to the various aggregate morphologies can be distingu ished by FTIR analysis. (C) 2001 Academic Press.