Research

Thesis title: "Calorimeter at Atomic Resolution"

Thesis outline: Understanding the quantitative relationships between protein structural dynamics and molecular thermodynamics is crucial to understanding biomolecular behavior and several associated phenomena, such as allostery, protein-ligand binding affinities, enthalpy-entropy compensation, and cryptic binding sites in drug targets. While several methods to determine the static structure of the thermodynamically more stable state of biomolecules exist, such as X-ray crystallography and cryo-electron microscopy, they are largely incapable of providing a complete thermodynamic ensemble. Solution-state NMR spectroscopy, however, stands out in this regard, as it can determine the dynamic conformational ensemble for any given biomolecule. As such, it is poised to serve as a source of information for molecular thermodynamics within large biomolecules.

Several approaches have been proposed to interpret thermodynamic parameters from NMR relaxation data over the last four decades. However, they are mostly limited to probing a single species within a multimolecular complex, small distance ranges, and fast timescale motions and cannot account for correlated motions. The Nuclear Overhauser Effect (NOE) is the NMR proxy without these limitations: it is ensemble- and time-averaged, sensitive to molecular exchange and motions across timescales. It also provides a dense sampling of interactions acting on both protein and ligand.

The theoretical relationship between NOE cross-relaxation rates at different temperatures enables one to derive changes in enthalpy, entropy, and heat capacity associated with conformational changes for a given pair of atoms. Developing upon established NMR experiments, the aim is to obtain accurate NOE cross-relaxation rates with varying temperatures and obtain a thermodynamic stability curve corresponding to each NOE, thereby establishing a calorimeter at atomic resolution.

The proposed methodology would need to be validated against previously proposed methods that calculate the entropic contributions of different molecule parts. Our treatment of spin diffusion, which directly affects the accuracy of obtained NOEs, would also be validated using perdeuterated proteins. These experiments would be carried out on the WW domain, a signaling protein and a prototypical model for allostery. The final aim is to apply the obtained insights toward predicting protein stability and designing mutants with finely tuned thermodynamic properties.

This project proposes a proof-of-concept study for accurately measuring thermodynamic parameters at atomic resolution to understand biomolecular behavior better. Its expected impact is broad, improving our fundamental understanding of protein stability and allostery with applications in computer-assisted rational drug design and protein engineering.

Supervisor & Co-Mentor: Julien Orts

Back