Production of ribosome-released nascent proteins with optimal physical properties.

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.