Design of sensors to measure macromolecular crowding in vivo

MARÍA SOLEDAD LABANDA; PATRICIO CRAIG; FLORENCIA VALLE CARDAMA; JULIO CARAMELO

Sierra de la Ventana, Buenos Aires

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica; 2014

Sociedad Argentina de Biofísica

The interior of cells is characterized by a high content ofmacromolecules which occupy between 20 and 40% of the totalvolume. Due to the mutual impenetrability of particles, this volumefraction is unavailable to other molecules, producing a steric repulsionthat generates important kinetic and thermodynamic consequences onprocesses occurring in vivo. This makes macromolecular crowding aphysiological parameter of great relevance that should be consideredduring in vitro experiments. The aim of this work is to develop a probeto measure macromolecular crowding in vivo. We started from achimeric protein consisting of two fluorescent proteins (CFP and YFP)linked by a natively unfolded linker, in which intramolecularfluorescence resonance energy transfer (FRET) can be induced. Toanalyze the effects of crowding on FRET efficiency of the protein, wemeasured fluorescence spectra of the protein in media with increasingconcentrations of different crowders (PEG, ficoll, BSA). Interestingly,FRET efficiency increases as crowder concentration increases, showing asharp and cooperative change. In order to understand theseobservations, we performed coarse grained molecular dynamicssimulations of the protein at various fractions of volume occupied by aninert crowding agent. The analysis of the trajectories shows that theaverage distance between chromophores decreases as crowding levelincreases. These results suggest that in crowded conditions the proteinadopts compact conformations, decreasing the distance between donorand acceptor, and hence, increasing FRET.