Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy.

It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate state prior to aggregation. Based on a single real-time 2D (13)C-(13)C transition spectrum, kinetic information about the refolding and aggregation step could be extracted. In addition, structural rearrangements associated with refolding are estimated and several different aggregation scenarios were compared to the experimental data.