title: Production of ribosome-released nascent proteins with optimal physical properties.
abstract: The growing interest in protein folding under physiologically relevant conditions has prompted investigations requiring direct comparisons between ribosome-bound and ribosome-released nascent proteins. Such studies, involving the ad hoc release of newly synthesized proteins from stalled ribosomes, demand a release agent able to produce nonaggregated native proteins and preserve the overall nature of the medium. Here, we explore hydroxylamine, a reactant rarely used to release nascent chains, and compare it to other ribosome-release agents: puromycin, RNase A/EDTA, and sodium hydroxide. Ribosome-bound nascent chains corresponding to the sequence of apoHmpH, the Escherichia coli N-terminal domain of Hmp, were used as a model system. Fluorescence anisotropy decays were employed to probe the self-association and overall physical properties of nascent proteins. Gel electrophoresis and RNA chip microfluidic capillary electrophoresis yielded information on the integrity of nascent peptidyl-tRNAs and ribosomes, respectively. Of the four reagents examined, only hydroxylamine releases nascent apoHmpH without causing extensive aggregation or degradation of the ribosome. Hydroxylamine does not introduce large hydrophobic C-terminal modifications and functions at nearly physiological pH. It is therefore a suitable reagent for the ad hoc release of nascent proteins from the ribosome.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Ziehr
authors.forename: Jamie P
authors.initials: JP
authors.lastname: Ellis
authors.forename: Peter H
authors.initials: PH
authors.lastname: Culviner
authors.forename: Silvia
authors.initials: S
authors.lastname: Cavagnero
mesh_terms: D004492:Edetic Acid / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D005454:Fluorescence Polarization; D019811:Hydroxylamine / Q000494:pharmacology; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D055585:Physical Phenomena; D014176:Protein Biosynthesis / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000378:metabolism*; D012972:Sodium Hydroxide / Q000494:pharmacology; D014461:Ultracentrifugation
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D019811:Hydroxylamine; D011691:Puromycin; D012972:Sodium Hydroxide; D004492:Edetic Acid; D012259:Ribonuclease, Pancreatic