Grant: Research Tools and Instruments

Proteins fold into complex structures that are required for correct function. These structures involve frustrated' interactions that can't always be satisfied, allowing alternative structures that cause dysfunction to form. Indeed, many diseases are linked to aggregates of such misfolded proteins. Understanding how aggregation competes with native folding will yield key insights into how folding goes wrong and improved principles for designing new proteins. We will probe aggregation using interferometric light scattering to measure the mass of single proteins in solution. Because this method is sensitive to single molecules, it can monitor the earliest steps of aggregationsmall oligomerswithin a heterogeneous mixture covering all stages of the aggregation cascade; because it is label-free, aggregation can be probed without any bias from the fluorescent dyes normally used for detection. We will use single-molecule (SM) mass photometry to distinguish oligomers of different size (dimer, trimer,) and catalog their formation/growth rates, obtaining for the first time an unbiased, quantitative measure of each state in the aggregation cascade for comparison to physical models. We will probe 2 specific aims:***1. How proteins switch from native to aggregated when conditions change. We will focus on the tau protein as an example: it normally helps stabilize microtubules in neurons, but switches to aggregate in Alzheimer's disease and other dementias. We will compare the formation/growth rates of oligomers under normal and diseased conditions, fitting to microscopic models to deduce the mechanism driving the change in behaviour.***2. How evolution shapes the competition between native folding and aggregation. Evolution is thought to select for smooth energy landscapes that allow rapid formation of minimally frustrated structures. To study this question, we will (A) compare aggregation of ancestral proteins from different evolutionary periods, focusing on PrP, a protein that aggregates to cause disease and whose sequence we have reconstructed back to ~350 million years ago; and (B) measure aggregation of proteins designed computationally, which have not undergone evolutionary selection and are often prone to aggregation even though de novo design is increasingly reliable. In each case, we will compare the formation/growth rate of oligomers measured by SM mass photometry to properties of the native folding like stability, barrier height, and landscape roughness that we are measure using SM force spectroscopy, to identify the physical changes in the folding related to differences in aggregation-propensity.***Such studies of aggregation with SM sensitivity, not possible until the recent development of SM mass photometry, present a timely and exceptional opportunity to make significant advances in a decades-old problem with relevance to biology, physics, chemistry, and medicine. Without this instrumentation, we will not be able to pursue such exciting science.