The goal of this project is to develop a novel and highly sensitive approach to monitor structural and dynamic parameters associated with the co-translational folding and mis-folding of nascent polypeptide chains. Intra and inter-molecular distances and distance distributions between different amino acids within ribosome bound nascent chains will be followed by conventional fluorescence resonance energy transfer (FRET) and by lanthanide-based fluorescence resonance energy transfer (LRET) techniques in cell-free systems. Very little is currently known on how polypeptide structure evolves during translation, yet this issue is of key importance in understanding how proteins actually fold (and misfold) in the cell. This is a very significant issue in biology, since the sequence-structure paradigm has never been directly explored in the natural milieu. This research also promises to yield useful information leading to a better understanding of the structural and temporal bases of mis-folding, including the possible role played in nature by cotranslational self-association, possibly leading to co-translational mis-folding. The following specific aims will be pursued: (1) Implementation of novel tools to monitor inter- and intra-molecular distances in ribosome-bound nascent polypeptides. The above tools include: (a) efficient generation of monodisperse ribosome-bound nascent polypeptides; (b) preparation of a few selected donor- and acceptor-labeled aminoacyl-tRNAs; (c) testing the co-translational incorporation of these constructs and (d) FRET and LRET measurements in cell-free systems. (2) Utilization of the tools developed within Specific Aim 1 to measure inter- and intra-molecular distances in nascent polypeptide chains of different predetermined length by FRET and LRET in cell-free systems. Two model proteins which express efficiently in E. coli (chloramphenicol acetyltransferase and apomyoglobin) will be targeted for this work. The feasibility of this research plan is supported by our documented ability to generate virtually monodisperse ribosome-bound nascent chains of predetermined length, and by the fact that preparation of fluorophore-labeled amino acyl-tRNAs as well as FRET/LRET measurements in cell-like environments are widely documented in the literature.
|Li, Fuyang; Dong, Junachao; Eichmiller, Robin et al. (2013) Role of Saw1 in Rad1/Rad10 complex assembly at recombination intermediates in budding yeast. EMBO J 32:461-72|
|Fedyukina, Daria V; Cavagnero, Silvia (2011) Protein folding at the exit tunnel. Annu Rev Biophys 40:337-59|
|Ellis, Jamie P; Culviner, Peter H; Cavagnero, Silvia (2009) Confined dynamics of a ribosome-bound nascent globin: Cone angle analysis of fluorescence depolarization decays in the presence of two local motions. Protein Sci 18:2003-15|
|Ellis, Jamie P; Bakke, Courtney K; Kirchdoerfer, Robert N et al. (2008) Chain dynamics of nascent polypeptides emerging from the ribosome. ACS Chem Biol 3:555-66|
|Kirchdoerfer, Robert N; Huang, Joseph J-T; Isola, Molly K et al. (2007) Fluorescence-based analysis of aminoacyl- and peptidyl-tRNA by low-pH sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 364:92-4|