Structure and interaction of enzyme networks and their metabolites in situ by Raman microspectroscopy

Catalysis spans different scales: from activating particular bonds over catalytic cascades to the embedding of catalytic systems in larger environments, such as cells. Organising catalysts in subcellular niches leads to advantages regarding concentrations and optimised microenvironments, but also to questions such as how are trafficking and local coordination achieved. This concerns catalysts (enzymes) but also substrates and products. An additional layer of complexity is added through the modulation of catalysis in response to cellular needs. Catalysis as an integrated process comprising catalytic systems, modulators and logistics is becoming studiable through in situ methods such as crosslinking and in situ spectroscopic methods. They allow the detection of protein-protein interactions in their natural context and may reveal the metabolic response to system challenges by information on enzyme and/or metabolite structure and interaction.
The project will uncover enzyme networks de novo, connect them with networks of metabolites, and characterize the underlying structure and composition of catalysts in these specific cascades in situ. We will obtain information on enzymes operating in selected organelles, such as mitochondria or the endolysosomal environment. These compartments can be observed in intact cells, but they can also be identified in isolated fractions, and they can be purified and fractionated and serve as source of ‘pure’ enzyme-networks. The expertise of the team members in protein crosslinking and the description of protein-protein interaction using in situ vibrational spectroscopy and microscopy at micron to nanoscale resolution, as well as NMR spectroscopy of proteins in membranes will be complemented by theoretical work that can describe protein interaction in real local environments. We will study the metabolic response of cultured cells to specific challenges, e.g., starvation, pH change etc. at the molecular level (enzyme structure and interaction and substrate / product) in situ. The combination of protein-protein and protein-substrate/product composition and interaction (molecular structural information) with the verification of the actual involvement of particular enzymes in networks by crosslinking, supported by MD simulations that take into account crowding and the formation and disruption of interactions is unique from the methodological perspective. It will connect understanding of enzyme catalysis at the molecular level with localization of the processes in the real biosystem or other complex environment.