Practical Synthesis of -Formylmethionylated Peptidyl-tRNA Mimics.

Publication Type:

Journal Article

Source:

ACS Chem Biol (2023)

Abstract:

<p>Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are frequently needed for structural and functional studies of protein synthesis in the ribosome. Such conjugates are accessible by chemical solid-phase synthesis, allowing for the utmost flexibility of both the peptide and the RNA sequence. Commonly used protection group strategies, however, have severe limitations with respect to generating the characteristic -formylmethionyl terminus because the formyl group of the conjugate synthesized at the solid support is easily cleaved during the final basic deprotection/release step. In this study, we demonstrate a simple solution to the problem by coupling appropriately activated -formyl methionine to the fully deprotected conjugate. The structural integrity of the obtained -formylmethionyl conjugate─and hence the chemoselectivity of the reaction─were verified by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry sequence analysis. Additionally, we confirmed the applicability of our procedure for structural studies by obtaining two structures of the ribosome in complex with either fMAI-nh-ACCA or fMFI-nh-ACCA in the P site and ACC-PMN in the A site of the bacterial ribosome at 2.65 and 2.60 Å resolution, respectively. In summary, our approach for hydrolysis-resistant -formylated RNA-peptide conjugates is synthetically straightforward and opens up new avenues to explore ribosomal translation with high-precision substrate mimics.</p>

PDB: 
Coordinates and structure factors were deposited in the RCSB Protein Data Bank with accession codes 8T8B for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn, and P-site peptidyl-tRNA analog fMAI-nh-ACCA; 8T8C for the T. thermophilus 70S ribosome in complex with protein Y, A-site aminoacyl-tRNA analog ACC-Pmn, and P-site peptidyl-tRNA analog fMAI-nh-ACCA
Detector: 
EIGER2
Beamline: 
24-ID-C
24-ID-E