Single-molecule tracking for live-cell protein synthesis kinetics

Descripción del proyecto

Revelación de los determinantes moleculares de la síntesis de proteínas «in vivo»

La síntesis, el direccionamiento y el plegamiento correctos de las proteínas son primordiales para todas las células vivas. Dado que hay una demanda creciente de proteínas terapéuticas como los anticuerpos, existe una necesidad urgente de comprender y controlar los mecanismos moleculares responsables de este proceso. El proyecto SMACK, financiado con fondos europeos, investigará cómo la secuencia del ARNm determina en última instancia cómo, dónde y cuándo se pliega un polipéptido. Mediante un sistema experimental y analítico basado en fluorescencia, los científicos analizarán la cinética de la síntesis de proteínas a una escala monomolecular directamente dentro de la célula viva, así como de qué modo los antibióticos clínicamente pertinentes inhiben este proceso. Los conocimientos obtenidos ofrecerán pistas sobre el diseño de los sistemas de producción de proteínas de base microbiana, así como sobre el diseño de nuevos antibióticos.

Objetivo

This project aims to map the determinants of efficient synthesis, folding, and targeting of proteins in living bacterial cells, and to understand how antibiotic drugs inhibit these events. Our findings will be used to form a quantitative description of how the mRNA sequence ultimately determines how, where, and when a polypeptide should fold, which will help to design better E. coli-based production systems for recombinant proteins; both protein folding and targeting are currently severe bottlenecks. Further, by studying the effect of clinically relevant antibiotic drugs directly on ongoing protein synthesis, we will learn how the inhibitory action on the molecular level is translated into the response on cell and population level. This knowledge will help to improve the use of current drugs, as well as to provide clues regarding how new drugs should be designed.

To reach our goals, we will use and further develop our recently published fluorescence-based experimental and analytical system for live-cell single-molecule analysis of protein synthesis kinetics. Particularly, we will develop orthogonal translation systems to measure protein synthesis kinetics on defined mRNAs in an unperturbed cell background. We will also label and study key components of the universal pathway for Signal Recognition Particle-mediated cotranslational targeting of peptides to the membrane-bound peptide translocation complexes. In these applications we use small organic dyes for site-specific labeling of molecules, which allow recording of long diffusion trajectories inside cells. These trajectories are then fed to our machine-learning algorithms to deduce the binding kinetics.

With an increasing demand for therapeutic proteins, such as monoclonal antibodies for cancer treatment, there is an urgent need for microbial-based systems for cost-effective production of recombinant proteins. With our unique experimental and analytical system, we have the opportunity to aid in this development.