Functional effects of condensates on enzyme dynamics in the nucleus

We will use this award to uncover how enzymes change shape inside a cellular body known as a biomolecular condensate, both in a test tube and in cells. Despite attracting a vast amount of attention recently, how these condensates affect enzymes remains a mystery, due to their complexity.

For cells to function efficiently, they need to organise themselves to ensure essential molecules are present in the right place at the right time. Large eukaryotes achieve this by forming compartments enclosed inside plasma membranes. However, processes can also be organised in time and space by selectively concentrating components in membraneless biomolecular condensates, using a process known as liquid-liquid phase separation (LLPS). The last ~15 years have seen an explosion of interest in how biomolecular condensates regulate cellular processes, but despite intense research, large questions and controversies remain. In particular, we don't understand how condensates affect the different shapes enzymes adopt, and how they work. Moreover, we still don't understand if proteins behave the same way inside condensates in the complex and crowded cellular environment, as they do in the test tube; it has always been difficult to accurately replicate the complex environment of proteins, nucleic acids and other structures that are packed inside a cell in the test tube. Here, we will look at how enzymes change shape, and ask how this is affected by being squeezed into the dense environment inside a condensate: Does this fundamentally change the enzymes' ability to move, or does the enzyme carry on as normal?

To answer this we need a model that maintains the complexity necessary to form condensates in cells, but which allows precise experiments in the test tube. Our target enzyme, CBP, works inside condensates that normally form inside cells to turn genes on and off, giving us a tractable system that keeps all of the complexity needed for biomolecular condensates to form.

We also need a way to watch how enzymes change shape. We will bind two different colour fluorescent dyes to our target protein; when one of these dyes is switched on using a laser, it transfers some of its energy to the neighbouring dye, causing that molecule to glow. When applied to individual molecules, this is known as single-molecule Fluorescence Resonance Energy Transfer (smFRET). Importantly, smFRET is incredibly sensitive to how close together the two dyes are, so we can use it to watch protein movements as they happen. Such measurements are impossible when a group of molecules is studied, as the movements average out. Excitingly, we can do the same smFRET experiments in both the test tube and inside cells, to find out whether condensates affect proteins the same way in both models. To use smFRET we need to get fluorescent dyes into our target protein. To do this, we will hijack the machinery responsible for building proteins in the cell - the translation machinery - in a process called Genetic Code Expansion (GCE), where we add an extra amino acid to those normally used to build proteins. This extra amino acid can be fluorescent, and incorporated anywhere in our target protein, allowing us to label different parts of a protein to study movement.

The Pioneer Award will provide an unprecedented opportunity to alter current models of condensate function. LLPS is still a controversial subject due to the lack of convincing mechanisms for condensate function. By watching how squeezing enzymes into a crowded condensate alters their ability to change shape, we will provide a definitive mechanism for function, providing a new framework for looking at mechanism and causing a paradigm shift in current thinking.