The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.

Multiple site-selective insertions of noncanonical amino acids into sequence-repetitive polypeptides.

A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pair and the noncanonical amino acid. Elastin-mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein-based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.