Quantification and consequences of macromolecular crowding in living cells

Cells are highly crowded with proteins and polynucleotides, with concentrations ranging from 80 to 400 mg/mL. Knowledge of crowding is critical to understand cell physiology and to assess its relevance for medical science and biotechnology: Any reaction that depends on the available volume can be affected by crowding, including biopolymer diffusion, conformation, association, folding, phase separation, and aggregation. However, the effects of crowding in cells are complex and have remained difficult to tract. The applicant recently developed the first sensor to quantify the macromolecular crowding in living cells. This sensor is an excellent platform to determine the consequences of macromolecular crowding in living cells. The aim of this proposal is to quantify the consequences of cellular crowding in space and time, using model proteins as sensors inside living cells. The aim of the project will be pursued through the following objectives: 1. To establish the relation between the structure of a protein and the intracellular crowding. By mimicry of multi-domain proteins with engineered FRET-based crowding sensors, I will determine how the steric and chemical components of crowding influence the structural features of proteins in living cells. Systematic structural variations, supported by theory and simulation, will allow quantification of the different crowding effects. 2. To determine the role of crowding in the cell by perturbation of crowding and monitoring subsequent spatiotemporal variations. To understand the role of crowding for cells we will perturb this physical chemical property in vivo. Possible sources of crowding variation will be identified and the corresponding effects on proteins will be quantified, and possible health compromising consequences for the cell will be characterized. Knowledge of crowding inside living cells allows understanding of the behavior of biomolecules under physiological conditions and provides a framework to study the pathology of protein misfolding and aggregation.

Further Information

Assoziierter Wissenschaftler, Utrecht University

Dr. Arnold Boersma

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Theodoris Pittas, M.Sc.

+49 241 80-20658
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