Design of sensors to measure macromolecular crowding in vivo

LABANDA MS; CRAIG PO; CARAMELO, JJ

Villa Carlos Paz

Congreso; Biophysical Society of Argentina 42th Annual Meeting; 2013

Sociedad Argentina de Biofísica (SAB)

The interior of cells is characterized by a high content of macromolecules which occupy between 20 and 40% of the total volume1. Due to the mutual impenetrability of particles, this volume fraction is unavailable to other molecules, producing a steric repulsion that generates important kinetic and thermodynamic consequences on processes occurring in vivo2. This makes macromolecular crowding a physiological parameter of great relevance that should be considered during in vitro experiments. The aim of this work is to develop a probe to measure macromolecular crowding in vivo. We started from a chimeric protein consisting of two fluorescent proteins (CFP and YFP) linked by a natively unfolded linker, in which intramolecular fluorescence resonance energy transfer (FRET) can be induced3. To analyze the effects of crowding on FRET efficiency of the protein, we measured fluorescence spectra of the protein in media with increasing concentrations of PEG 8000. Interestingly, FRET efficiency increases as PEG concentration increases, showing a sharp and cooperative change. In order to understand these observations, we performed coarse grained molecular dynamics simulations of the protein at various fractions of volume occupied by an inert crowding agent. The analysis of the trajectories shows that the average distance between chromophores decreases as crowding level increases. These results suggest that in crowded conditions the protein adopts compact conformations, decreasing the distance between donor and acceptor, and hence, increasing FRET.