Research Projects

The Anfinsen dogma states that a protein’s amino acid sequence determines its unique, stable native structure under physiological conditions. Yet proteins are intrinsically dynamic, sampling an ensemble of conformations. While structural studies capture stable ground‑state forms, the low-population, short-lived excited states—often invisible in static snapshots—frequently represent the protein’s functional form, driving activities like catalysis, allostery, and signaling . Understanding protein function thus requires mapping the full energy landscape, including the structure, thermodynamics, and kinetics of these excited states. Over evolutionary timescales, natural selection has optimised not only the ground-state structures but also the energetic and dynamic properties of excited states . Ancestral Sequence Reconstruction (ASR) enables the resurrection of ancient proteins to experimentally probe how mutations reshaped their conformational landscapes. A prime example is lymphotactin (XCL1), a metamorphic chemokine that exists in equilibrium between two native structures: a monomeric chemokine fold that signals via XCR1, and a dimeric β-sheet fold that binds glycosaminoglycans. Dishman et al. applied ASR to resurrect the ancestral variants of XCL1, namely Anc.0, Anc.1, Anc.2, Anc,3 and Anc.4 and used Nuclear Magnetic Resonance spectroscopy to understand the evolutionary emergence of metamorphosis in XCL1. The resurrection of ancestral XCL1 proteins has revealed that metamorphosis was selected for during evolution and is not merely ‘evolution caught in action’. Their results show how the variants exhibited progressive acquisition of metamorphic behavior through mutations affecting disulfide bonds, dimer interfaces, and intramolecular contacts in the chemokine fold, culminating in the extant form of XCL1. Despite these structural insights, the role of excited-state dynamics in the evolution of XCL1 remains unexplored. We hypothesize that specific excited states in ancestral variants have been thermodynamically and/or kinetically stabilized, facilitating the modern ~50:50 fold equilibrium in XCL1. We propose to characterise and compare the excited-state populations and lifetimes in XCL1 and Anc.0–Anc.4 using advanced NMR techniques including CPMG- and CEST- based experiments. By elucidating how excited-state landscapes evolved, this study will illuminate fundamental links between sequence, structure, dynamics, function, and evolution. These insights will not only expand our understanding of protein metamorphism but also guide future efforts in protein engineering and evolutionary biophysics.