Protein dynamics, stability and enzymology

We currently work on four different projects:

• Gaussia Luciferase
• Marine Bioluminescence
• Protein engineering

Gaussia luciferase – a highly dynamic enzyme

Our principal model system is Gaussia luciferase (GLuc), a small and exceptionally bright bioluminescent enzyme from the marine copepod Gaussia princeps. GLuc catalyzes oxidation of the small molecule coelenterazine (CTZ), producing blue light.

GLuc is unusual in several respects. Despite its high catalytic activity, our work has shown that the protein has surprisingly little of the well-packed hydrophobic core normally associated with a stable enzyme structure and displays extensive conformational dynamics. This makes GLuc an interesting model for exploring how a highly dynamic protein can nevertheless perform efficient and highly specific catalysis.

We are particularly interested in the conformational changes accompanying substrate binding and catalysis. Structural and mutational studies have identified residues that are essential for light production, but their positions in substrate-free structures are difficult to reconcile with a conventional, pre-organized active site. Our current work therefore investigates how binding of CTZ reorganizes the conformational ensemble of GLuc and generates the catalytically active state.

Another unusual property of GLuc is its characteristic flash kinetics. The enzyme initially produces an intense burst of light but rapidly loses activity. We have shown that this behavior results from progressive, reaction-dependent inactivation of the enzyme, associated with covalent modification by compounds generated during the reaction. We are now investigating how catalysis, conformational dynamics and this self-inactivation process are connected.

Together, these properties make GLuc a useful experimental system for addressing a broader question: how can a dynamic ensemble of protein conformations generate a precise catalytic function?

Marine bioluminescence and the origin of coelenterazine

Our interest in GLuc has also led us to investigate the broader biology of marine bioluminescence. Coelenterazine is used as a luciferin by many unrelated marine organisms, yet surprisingly little is known about how it is synthesized in nature.

We are investigating the biosynthetic origin of coelenterazine, particularly in the bioluminescent comb jelly Mnemiopsis leidyi. This work extends our interest from the molecular mechanism of a luciferase to the biological systems responsible for producing its substrate and ultimately to the evolution of marine bioluminescence.

Protein engineering and sequence–function relationships

A complementary theme in our research is understanding how protein sequence determines structure, dynamics and function. We use mutagenesis, genetic selection and quantitative biochemical assays to explore sequence–function relationships and to identify protein variants with altered properties.

Rather than viewing protein engineering solely as a means of producing improved proteins, we use it as an experimental tool for understanding proteins: systematic changes in sequence can reveal which structural and dynamic features are required for activity and which can be modified or replaced.