Uncovering how chaperones assist in protein folding and preventing toxic protein aggregates - Project Summary/Abstract Proteins carry out the majority of cellular functions and are essential for cellular activities. Most proteins must fold into the correct shape to fulfill their functions and need to endure constantly changing conditions within the cell. However, proteins cannot accomplish this on their own; they rely heavily on chaperones. Different chaperones fulfill various functions to ensure proteostasis. Here, we focus on Hsp60 and Hsp10, which assist in protein folding and prevent protein aggregation. Protein misfolding is implicated in many severe diseases such as Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, cystic fibrosis, and Huntington's disease. Despite decades of research, there is a fundamental gap in understanding the molecular mechanisms underlying the protective role of chaperones. This proposal will address four key questions: 1. How does ATP induce a simultaneous conformational change in all subunits of Hsp60? ATP can bind to each subunit of the heptameric or tetradecameric Hsp60 inducing an enormous conformational change. It is a cooperative process, but how the different subunits communicate with each other remains unknown. Understanding this allosteric mechanism is key to comprehending the kinetic cycle of Hsp60, which involves native substrate binding, encapsulation, and release of the folded substrate. 2. How do substrates interact with a chaperone? Is there a common binding motif? What is the timescale? A given chaperone can interact with many different client proteins. This study aims to determine if there is a common binding motif for substrates when interacting with the chaperone. This will provide detailed molecular principles of how substrates interact with chaperones, and this knowledge is valuable to provide insights into the chaperone's selectivity and its ability to handle various types of substrates. 3. What is the purpose of the tails of Hsp60? Several chaperones have unstructured flanking regions that are essential for their function. However, due to their flexible nature, these regions are not easily accessible to most biochemical methods. Solution-state NMR can provide visualization of how these regions interact with substrates, unraveling their contribution to substrate binding and encapsulation. 4. What is the role of the co-chaperone, Hsp10? Hsp10 has been overlooked for many decades, thought to be merely the helper of Hsp60. Recent studies reveal that Hsp10, in the absence of Hsp60, assists in protein folding and inhibits protein aggregation. Studies, how Hsp10 in the absence of Hsp60 interacts with substrates will help understand how Hsp10 contributes to proteostasis. The combination of all these studies will shed light on fundamental questions about the mechanisms that chaperones and co-chaperones use to efficiently fold proteins into their functional forms and prevent aggregation. A process which crucial to maintaining protein homeostasis, which is vital for life.
