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Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

ABSTRACT: The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC-ribosome contact affecting translational accuracy. We identified a local contact point of RAC to the ribosome. The data provide the first experimental evidence for the existence of a four-helix bundle as well as a long α-helix in full-length RAC, in solution as well as on the ribosome. Additionally, we complemented the structural picture of the region mediating this functionally important contact on the 40S ribosomal subunit. In sum, this study constitutes the first application of SDSL-EPR spectroscopy to elucidate the molecular details of the interaction between the 3.3 MDa translation machinery and a chaperone complex.

SUBMITTER: Fries SJ

PROVIDER: S-EPMC8060413 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

Fries Sandra J SJ Braun Theresa S TS Globisch Christoph C Peter Christine C Drescher Malte M Deuerling Elke E

Scientific reports 20210421 1

The eukaryotic ribosome-associated complex (RAC) plays a significant role in de novo protein folding. Its unique interaction with the ribosome, comprising contacts to both ribosomal subunits, suggests a RAC-mediated coordination between translation elongation and co-translational protein folding. Here, we apply electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labeling (SDSL) to gain deeper insights into a RAC-ribosome contact affecting translational accuracy. W ...[more]

PMID: 33883604

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Project description:The chemical interplay of nitrogen oxides (NO's) with hemoglobin (Hb) has attracted considerable recent attention because of its potential significance in the mechanism of NO-related vasoactivity regulated by Hb. An important theme of this interplay-redox coupling in adducts of heme iron and NO's-has sparked renewed interest in fundamental studies of FeNO(x) coordination complexes. In this Article, we report combined UV-vis and comprehensive electron paramagnetic resonance (EPR) spectroscopic studies that address intriguing questions raised in recent studies of the structure and affinity of the nitrite ligand in complexes with Fe(III) in methemoglobin (metHb). EPR spectra of metHb/NO(2)(-) are found to exhibit a characteristic doubling in their sharper spectral features. Comparative EPR measurements at X- and S-band frequencies, and in D(2)O versus H(2)O, argue against the assignment of this splitting as hyperfine structure. Correlated changes in the EPR spectra with pH enable complete assignment of the spectrum as deriving from the overlap of two low-spin species with g values of 3.018, 2.122, 1.45 and 2.870, 2.304, 1.45 (values for samples at 20 K and pH 7.4 in phosphate-buffered saline). These g values are typical of g values found for other heme proteins with N-coordinated ligands in the binding pocket and are thus suggestive of N-nitro versus O-nitrito coordination. The positions and shapes of the spectral lines vary only slightly with temperature until motional averaging ensues at approximately 150 K. The pattern of motional averaging in the variable-temperature EPR spectra and EPR studies of Fe(III)NO(2)(-)/Fe(II)NO hybrids suggest that one of two species is present in both of the alpha and beta subunits, while the other is exclusive to the beta subunit. Our results also reconfirm that the affinity of nitrite for metHb is of millimolar magnitude, thereby making a direct role for nitrite in physiological hypoxic vasodilation difficult to justify.

Dataset Information

Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

Publications

Deciphering molecular details of the RAC-ribosome interaction by EPR spectroscopy.

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