Grants and Contributions:

Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023)

In the early 1960s, Anfinsen laid down the foundations of the dogma that has become the cornerstone of protein biochemistry: namely, that the native fold of a protein is determined by its amino acid sequence. However, this neglects the role that inter-domain interactions play in the folding of multi-domain proteins. Protein domains by definition are capable of folding into their functional folds independently, but the presence of neighboring domains must also affect the protein fold. The major aim of my research program is to provide a quantitative molecular-level description of: a) how inter-domain interactions contribute to the overall stability of multi-domain proteins; b) how these inter-domain interactions influence the protein folding mechanism. I propose to investigate the significance of these interactions by studying the folding of the enzyme Otu1 from the yeast S. cerevisiae as a paradigm multidomain enzyme. Otu1 is a protein containing 348 amino acids and catalyzes the cleaving of isopeptide bonds involving ubiquitins. This protein forms three distinct domains: the ubiquitin-like domain, the catalytic OTU domain, and the putative Zn-binding domain. Each of these three domains are capable of folding separately into their biologically active conformations. The research program for my group, that includes 4 HQP at any given time, will include the following steps.

1) First, study the thermodynamic properties of each domain by monitoring the thermal and chemical denaturation profile of each domain. These denaturation profiles can be monitored with the following instrumentation that exists in my laboratory: differential scanning calorimetery, circular dichroism spectroscopy and fluorescence steady-state.

2) Study the folding kinetics of each domain. Folding processes in proteins span a large number of timescales (microseconds to minutes), therefore we will study the kinetics of domain folding and unfolding using a combination of molecular biology and unique spectroscopy techniques. Judicious amino acid point mutations will be made in different protein locations so that we can locally probe protein folding in real time; while, my recently acquired state-of-the-art kinetic instrumentation (stopped flow, temperature jump spectroscopy and time-resolved fluorescence) allows us to access protein folding timescales spanning nanoseconds to minutes.

3) The combination of kinetic and thermodynamic data will provide us with a complete picture of the folding pathway of each domain. We will then use the protocols developed in parts 1 and 2 to study the folding kinetics and thermodynamics of the complete protein.

We shall thus quantify how domain-domain interactions influence protein stability and the folding process in the Otu1 system. These results will provide us with a blue print to extend our investigations to study the folding of other multi-domain enzymes in the future.