10.1021/ac902952b.s001
David R. Ziehr
David R.
Ziehr
Jamie P. Ellis
Jamie P.
Ellis
Peter H. Culviner
Peter H.
Culviner
Silvia Cavagnero
Silvia
Cavagnero
Production of Ribosome-Released Nascent Proteins with Optimal Physical Properties
American Chemical Society
2010
gel electrophoresis
protein
Fluorescence anisotropy
sodium hydroxide
ribosome
Such studies
model system
Optimal Physical PropertiesThe
release agent
RNA chip microfluidic capillary electrophoresis
hydroxylamine releases
2010-06-01 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Production_of_Ribosome_Released_Nascent_Proteins_with_Optimal_Physical_Properties/2765602
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 <i>ad hoc</i> 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 <i>Escherichia coli</i> 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 <i>ad hoc</i> release of nascent proteins from the ribosome.