A System of Minimalist Protein Labels for Fluorescence Studies

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

This project is jointly funded by the Chemistry of Life Processes Program in the Division of Chemistry and the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences. The Chemistry of Life Sciences Program and the Molecular Biophysics Cluster fund Professor E. James Petersson of the University of Pennsylvania to develop tools to study the molecular details of protein motions. In order to function, proteins must fold into the correct shape and with the proper flexibility. Dynamic shapes are important to healthy cells, but folding into the wrong shape can cause a protein to become toxic to cells. The Petersson laboratory is developing a system of computational and experimental tools to study protein shape changes. The tools consist of fluorescent labels that can be incorporated concomitantly as proteins are made in cells. In addition, computational methods for predicting the best labeling sites are being developed. These computational tools are also assisting in producing and interpreting results that can be used to make movies of protein motions, which will be helpful to better understand protein shape in a dynamic environment. Importantly, both the labeling materials and computer programs are being made available to other laboratories via websites. The broader impacts of this work include training undergraduate and graduate students in a multidisciplinary laboratory environment. The training environment makes use of organic synthesis, physical chemistry as well as molecular and cellular biology to investigate biological phenomena. The project is also developing hands-on demonstrations to teach K-12 students about fluorescence and protein folding in health and disease. In order to make these popular activities more accessible, videos and downloadable materials are being made available. These materials allow K-12 teachers to carry out similar exercises with their students. Both the proposed research products and educational tools are designed to benefit a wide audience. The flexibility of proteins allows them to access a variety of folded structures. Shifts between these structures are used to transmit signals within cells, facilitate enzymatic catalysis, or even generate mechanical force in the case of motor proteins. Recently, much attention has focused on intrinsically disordered proteins. These proteins have no stable structures and yet play important functional roles in cell health and disease. The Petersson Laboratory is developing new fluorescent amino acids probes based upon small aromatic scaffolds. These probes are genetically incorporated into the peptide backbone and are minimally perturbing to protein folding. The project builds upon the principal investigator's discovery that thioamide substitutions of the peptide backbone can serve as small fluorescence quenchers. In this case, equally minimalist sidechain thioamide probes are generated and genetically encoded. Photophysical studies of the fluorescent amino acids and/or thioamide quenchers are used to pair them in appropriate sets. The pairs are used to probe conformational changes over all relevant distance ranges in proteins. These minimalist tools are particularly valuable in co-translational protein folding studies or labeling the interior of a folded protein. Such experiments are currently inaccessible to common post-translational fluorescent labeling strategies. The project is also creating computational tools to identify non-perturbing sites for label incorporation. A strategy for incorporating fluorescence data as conformational constraints in models of protein motion is being developed. The computational tools are implemented in the popular, open-source platform Rosetta, so that they can be accessed and modified by the protein research community.