Amyloid fibril formation kinetics of low-pH denatured bovine PI3K-SH3 monitored by three different NMR techniques

Introduction: Misfolding of amyloidogenic proteins is a molecular hallmark of neurodegenerative diseases in humans. A detailed understanding of the underlying molecular mechanisms is mandatory for developing innovative therapeutic approaches. The bovine PI3K-SH3 domain has been a model system for aggregation and fibril formation.Methods: We monitored the fibril formation kinetics of low pH-denatured recombinantly expressed [U-13C, 15N] labeled bovine PI3K-SH3 by a combination of solution NMR, high-resolution magic angle spinning (HR-MAS) NMR and solid-state NMR spectra. Solution NMR offers the highest sensitivity and, therefore, allows for the recording of two-dimensional NMR spectra with residue-specific resolution for individual time points of the time series. However, it can only follow the decay of the aggregating monomeric species. In solution NMR, aggregation occurs under quiescent experimental conditions. Solid-state NMR has lower sensitivity and allows only for the recording of one-dimensional spectra during the time series. Conversely, solid-state NMR is the only technique to detect disappearing monomers and aggregated species in the same sample by alternatingly recoding scalar coupling and dipolar coupling (CP)-based spectra. HR-MAS NMR is used here as a hybrid method bridging solution and solid-state NMR. In solid-state NMR and HR-MAS NMR the sample is agitated due to magic angle spinning.Results: Good agreement of the decay rate constants of monomeric SH3, measured by the three different NMR methods, is observed. Moderate MAS up to 8 kHz seems to influence the aggregation kinetics of seeded fibril formation only slightly. Therefore, under sufficient seeding (1% seeds used here), quiescent conditions (solution NMR), and agitated conditions deliver similar results, arguing against primary nucleation induced by MAS as a major contributor. Using solid-state NMR, we find that the amount of disappeared monomer corresponds approximately to the amount of aggregated species under the applied experimental conditions (250 mu M PI3K-SH3, pH 2.5, 298 K, 1% seeds) and within the experimental error range. Data can be fitted by simple mono-exponential conversion kinetics, with lifetimes tau in the 14-38 h range. Atomic force microscopy confirms that fibrils substantially grew in length during the aggregation experiment. This argues for fibril elongation as the dominant growth mechanism in fibril mass (followed by the CP-based solid-state NMR signal).Conclusion: We suggest a combined approach employing both solution NMR and solid-state NMR, back-to-back, on two aliquots of the same sample under seeding conditions as an additional approach to follow monomer depletion and growth of fibril mass simultaneously. Atomic force microscopy images confirm fibril elongation as a major contributor to the increase in fibril mass.

Automated summary

In this study, we investigated the fibril formation kinetics of bovine PI3K-SH3 using a combination of solution NMR, high-resolution magic angle spinning (HR-MAS) NMR, and solid-state NMR. Our results showed that fibril elongation is the main mechanism contributing to the increase in fibril mass. We propose a combined approach using both solution NMR and solid-state NMR to simultaneously track monomer depletion and fibril mass growth.