title: Nanosecond dynamics of calmodulin and ribosome-bound nascent chains studied by time-resolved fluorescence anisotropy.
abstract: We report a time-resolved fluorescence anisotropy study of ribosome-bound nascent chains (RNCs) of calmodulin (CaM), a prototypical member of the EF-hand family of calcium-sensing proteins. As shown in numerous studies, in vitro protein refolding can differ substantially from biosynthetic protein folding, which takes place cotranslationally and depends on the rate of polypeptide chain elongation. A challenge in this respect is to characterize the adopted conformations of nascent chains before their release from the ribosome. CaM RNCs (full-length, half-length, and first EF-hand only) were synthesized in vitro. All constructs contained a tetracysteine motif site-specifically incorporated in the first N-terminal helix; this motif is known to react with FlAsH, a biarsenic fluorescein derivative. As the dye is rotationally locked to this helix, we characterized the structural properties and folding states of polypeptide chains tethered to ribosomes and compared these with released chains. Importantly, we observed decelerated tumbling motions of ribosome-tethered and partially folded nascent chains, compared to released chains. This indicates a pronounced interaction between nascent chains and the ribosome surface, and might reflect chaperone activity of the ribosome.
authors.affiliation: Institute of Complex Systems (ICS-5): Molecular Biophysics, Research Centre Jülich, Wilhelm-Johnen Strasse, 52425 Jülich (Germany).
authors.forename: Paraskevas
authors.initials: P
authors.lastname: Lamprou
authors.forename: Daryan
authors.initials: D
authors.lastname: Kempe
authors.forename: Alexandros
authors.initials: A
authors.lastname: Katranidis
authors.forename: Georg
authors.initials: G
authors.lastname: Büldt
authors.forename: Jörg
authors.initials: J
authors.lastname: Fitter
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D016296:Mutagenesis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D002147:Calmodulin; D011994:Recombinant Proteins
keywords: biosynthesis; cotranslational protein folding; fluorescence anisotropy decay; nascent polypeptide chains; proteins