Research Projects

The structure-function paradigm has served as the cornerstone of structural biology for over five decades and postulates that a protein sequence codes for a unique tertiary and quaternary structure that performs a unique function. Metamorphic proteins represent dramatic violations of this paradigm and adopt two distinct native structures in the absence of ligands and cofactors. The two structures interconvert between each other and often perform different functions. Metamorphic proteins emphasize the structural plasticity of protein folds and provide a striking illustration for how a single static structure is not enough to fully explain protein function. Although the structures of the alternate folds of several metamorphic proteins such as lymphotactin and Mad2 have been solved, the mechanism of fold-switching remains poorly understood. This is because the intermediates facilitating the interconversion are likely to be sparsely and transiently populated, and therefore invisible to most biophysical techniques. The broad goal of this proposal is to use cutting-edge NMR strategies such as saturation transfer (CEST) and relaxation dispersion (CPMG) to detect and structurally characterize such excited states and evaluate their role in protein metamorphosis. The human chemokine lymphotactin (Ltn) will be used as a model metamorphic protein in this proposal because its structure, function and evolution have been thoroughly characterized. Ltn is central to the immune response and participates in antiviral and antitumor defence mechanisms in humans. Ltn switches between a canonical monomeric a/b chemokine fold (Ltn10) that binds its cognate GPCR (XCR1), and a novel all-b dimeric fold (Ltn40) that recognizes glycosaminoglycans. Importantly, we have already detected an excited state of lymphotactin that coexists with Ltn10 using CEST and CPMG NMR experiments. We hypothesize that this excited state is critical in facilitating the metamorphosis of Ltn. We will address this hypothesis using the following specific aims: 1. Determine the structure of the excited state of Ltn10 using multinuclear CEST and CPMG experiments in conjunction with chemical shift-based structural modelling 2. Rigidify Ltn10 using protein engineering guided by structure and evolution, and evaluate the response of the free energy landscape and the stability of the excited state to this perturbation 3. “Search” for the presence of this excited state in the free energy landscape of metamorphic counterpart Ltn40 to connect the kinetic pathways between the two end conformations The excited state structure of Ltn will provide valuable insights into the nascent molecular events underlying protein metamorphosis and lay the foundation for elucidating how metamorphosis evolved. Ltn is a naturally conformational switch that rearranges rapidly without misfolding and aggregating. Conformational switches have recently emerged as vital components of synthetic circuits for manipulating cellular function. A mechanistic understanding of Ltn fold-switching will inform design principles for ab initio development of efficient molecular switches and biosensors. Moreover, misfolding and aggregation are central themes in the pathophysiology of protein conformational disorders such as Alzheimer's and Parkinson’s diseases. Understanding how Ltn navigates the fold-switching pathway efficiently and reversibly may also be beneficial for development of targeted therapeutics for combating protein conformational disorders such as Alzheimer's and Parkinson’s diseases.