Ribosomal Proteins of Deinococcus radiodurans: Their Solvent Accessibility and Reactivity

Abstract

The structure of proteins in native ribosomes from Deinococcus radiodurans R1 was probed by S-methylthioacetimidate (SMTA) modification of amino groups. The extent of protein labeling was quantified using top down methods, and modified positions were identified using bottom up experiments. Each protein's reactivity was predicted by examination of the crystal structures of the D. radiodurans 50S subunit and the T. thermophilus HB8 30S subunit. The close phylogenetic relation between D. radiodurans and T. thermophilus allowed the evaluation of D. radiodurans small subunit protein reactivity by alignment of homologous sequences. As a result, we were able to observe and characterize the reactivity of all of D. radiodurans ribosomal proteins. The extent of protein amidination was well correlated with the solvent-exposed surface area of each protein and even better correlated with the number of visible lysine residues. Lysine residues that are in close contact with rRNA structural features or buried in protein tertiary structure are nonreactive with SMTA, while those that are surface exposed are modified. Crystallographic disorder and post-translational modifications lead to differences between the observed and predicted extents of reactivity. Comparison of unmodified and disassembled amidinated protein mixtures also shows great promise for the quality control of the proteomic sequences and has facilitated the identification of four sequencing errors in the ribosomal proteome of D. radiodurans R1.

Chemical Reactivity of Brome Mosaic Virus Capsid Protein

“…32–38 Lysines that are protected from SMTA modification are buried at the interface of protein-protein or protein-nucleic acid interactions, or are involved in ionic orhydrogen bonding interactions with other amino acid residues. 33–34,36,38 The average extent of modification of proteins shows excellent agreement with predictions based on crystal structures, indicating minimal effects of native biomolecular structure. 33–38 …”

“…The localized molecular interactions responsible for the differential reactivity of K64 and K130 are probably hydrogen bonds or salt bridges, as observed in previous experiments involving SMTA modification of bacterial ribosomal proteins. 34–36,38 Inspection of the BMV capsid protein crystal structure (PDB:1JS9) reveals that the ε-amino group of K64 (red) is within 4.4 Å of one of the carboxyl oxygens of E160 (orange). If allowance is made for conformational flexibility of lysine and glutamate side chains (Fig.…”

“…These observations are consistent with SMTA modifications having only minimal effects on the structure of prokaryotic ribosomes. 34–36 …”

Dynamics of Ribosomal Protein S1 on a Bacterial Ribosome with Cross-Linking and Mass Spectrometry

“…Finally, spectra that corresponded to cross-links with multiple possible linkage patterns were manually inspected, and exact linkages were proposed only when the two cross-linked residues could be defined by the observed fragmentation. C-terminal lysine residues produced by trypsin were not considered as potential linkage sites, because amidination is known to block tryptic cleavage (35, 36). …”

“…Purified 30S subunits either containing or not containing protein S1 were modified with SMTA (S-methylthioacetimidate) under conditions similar to those used in previous ribosome labeling experiments (Scheme S2) (35, 39, 40). Unlike with cross-linking, the conditions of this experiment lead to modification of all solvent accessible primary amines.…”