Cell free protein synthesis and co-translational protein foldingFitter
Protein synthesis is a fundamental cellular process, by which ribosomes decode genetic information and convert it into an amino acid sequence. This highly complex process is accomplished by the translational machinery (ribosomes), accessory proteins, tRNA , mRNA and various factors. The fact that protein synthesis and translation does not necessarily require cell integrity, but can also proceed in so called cell-free protein systems opens the door for comprehensive studies to obtain a deeper understanding of individual steps of the translation cycle and of the folding of de novo synthesized proteins. We made use of this potential by real-time monitoring the green fluorescence protein (GFP) biosynthesis on single molecule level. A suppression of protein release from the ribosome after synthesis and time resolved imaging allowed for the determination of individual GFP maturation times and for quantifying the fraction of active ribosomes in the cell-free reaction format. Since co-translational folding is of particular importance for multi-domain proteins, we are currently developing approaches to monitor this kind of folding with PGK constructs.
A. Katranidis, D. Atta, R. Schlesinger, K.H. Nierhaus, T. Choli-Papadopoulou, I. Gregor, M. Gerrits, G. Büldt and J. Fitter
Fast biosynthesis of GFP molecules - a single molecule fluorescence study
Angewandte Chemie Int. Edit., 48, 1758-1761, (2009)
P. Lamprou, D. Kempe, A. Katranidis, G. Büldt, and J. Fitter
Nanosecond Dynamics of Calmodulin and Ribosome-Bound Nascent Chains Studied by Time-Resolved Fluorescence Anisotropy .
ChemBioChem, 15, 977-985, (2014)