A new sensor to measure macromolecular crowding in vivo

MARÍA SOLEDAD LABANDA; PATRICIO CRAIG; JULIO CARAMELO

Carlos Paz

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica

The interior of cells is characterized by a high content of macromolecules which occupy between 20 and 40% of the total volume. 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 vivo. 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 a FRET pair, CFP and YFP, linked by a natively unfolded linker (CtCRT), which has a high content of acidic amino acids. We made two variants using monomeric or dimeric variants of CFP and YFP. Fluorescence spectra of the proteins in media with increasing concentrations of PEG 8000 show that FRET efficiency increases as PEG concentration increases, suggesting that in crowded conditions the protein adopts compact conformations. In order to understand these observations, we performed coarse grained molecular dynamics simulations of the protein at various fractions of volume occupied by inert spheres. The analysis of the trajectories shows that the average distance between chromophores decreases as crowding level increases. Since the length and chemical identity of the unstructured linker may influence the behavior of the sensors, we are working on the replacement of the charged CtCRT linker by an inert and unstructured poly-glycine segment.