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Review
. 2019 Mar 14;20(6):1297.
doi: 10.3390/ijms20061297.

The Inescapable Effects of Ribosomes on In-Cell NMR Spectroscopy and the Implications for Regulation of Biological Activity

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Free PMC article
Review

The Inescapable Effects of Ribosomes on In-Cell NMR Spectroscopy and the Implications for Regulation of Biological Activity

David S Burz et al. Int J Mol Sci. .
Free PMC article

Abstract

The effects of RNA on in-cell NMR spectroscopy and ribosomes on the kinetic activity of several metabolic enzymes are reviewed. Quinary interactions between labelled target proteins and RNA broaden in-cell NMR spectra yielding apparent megadalton molecular weights in-cell. The in-cell spectra can be resolved by using cross relaxation-induced polarization transfer (CRINEPT), heteronuclear multiple quantum coherence (HMQC), transverse relaxation-optimized, NMR spectroscopy (TROSY). The effect is reproduced in vitro by using reconstituted total cellular RNA and purified ribosome preparations. Furthermore, ribosomal binding antibiotics alter protein quinary structure through protein-ribosome and protein-mRNA-ribosome interactions. The quinary interactions of Adenylate kinase, Thymidylate synthase and Dihydrofolate reductase alter kinetic properties of the enzymes. The results demonstrate that ribosomes may specifically contribute to the regulation of biological activity.

Keywords: Adenylate kinase; Dihydrofolate reductase; NMR spectroscopy; Ribosome; Thioredoxin; Thymidylate synthase; cross-correlated relaxation; enzyme activity; enzyme kinetics; mRNA; protein interactions; protein structure-function; rRNA.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Total cellular RNA alters in vitro spectra of Ubiquitin, Ubq. (A) In-cell 1H-15N heteronuclear single quantum coherence, HSQC, NMR spectra of [U- 15N] Ubq in P. pastoris after 24 h of methanol induction and (B) 48 h of methanol induction. (C) Overlay of the in vitro 1H-15N HSQC spectra of 10 μM [U- 15N] Ubq in the absence (black) and presence (red) of 30 mg/mL of RNABMM and (D) 30 mg/mL of RNABMDM. Insets in panel C show a broadening of selected residues of free Ubq (black) due to the interaction with RNABMM (red). (E) RNA from yeast cells grown with methanol, RNABMM, contains an amount of pre-mRNA and pre-rRNA larger than that of RNA from cells grown with a methanol/dextrose carbon source, RNABMDM. DNA MW indicates molecular weight markers. The numbers in panels A, B and C indicate some of the peak assignments. Panels A and B are adapted from Bertrand et al. (2012) [40]. Panels C, D and E are adapted from Majumder et al. (2016) [35].
Figure 2
Figure 2
1H-15N CRINEPT-HMQC-TROSY improves in-cell NMR spectral resolution. (A) Lysate HSQC spectrum of [U- 15N] Adenylate kinase, ADK. (B) In-cell 1H-15N HSQC spectrum of [U- 15N] ADK overexpressed for 16–18 h. (C) In-cell 1H-15N CRINEPT-HMQC-TROSY spectrum of [U- 2H, 15N] ADK overexpressed for 16–18 h. (D) In vitro 1H-15N CRINEPT-HMQC-TROSY spectrum of 10 µM purified [U- 2H, 15N] ADK in the presence of 2.5 µM ribosomes. The peak shapes in C and D arise from a population of free and bound species due to the high concentration of target protein (>100 µM).
Figure 3
Figure 3
Optimizing the CRINEPT transfer delay time yields in-cell target protein apparent molecular weights. (A) The dependence of Topt on the apparent in-cell molecular weight, MWapp at 700 MHz. Topt was experimentally determined at 5 °C (red symbols) by using 100 μM [U- 2H, 15N] Trx dissolved in 10 mM potassium phosphate buffer, pH 6.5, containing 30, 65, 75, and 85% (w/w) d5-glycerol with corresponding viscosities of 4, 34, 92, and 343 cP, respectively [56]. The MWapp of E. coli ADK and Trx in-cell and in vitro in the presence of total E. coli RNA, uncorrected for intracellular viscosity, are indicated. (B,C) The relative volumes of the G32, K141 and E162 peaks in the 1H-15N CRINEPT-HMQC--TROSY spectra of [U- 2H, 15N] ADK collected in-cell (B) and in vitro at 20 µM in the presence of 50 µg of total RNA (C) are plotted against CRINEPT transfer delay times. In (B) an in-cell value of 1.2 ms was assigned because shorter transfer delay times interfere with CRINEPT pulses and limit the ability to acquire data. An endogenous tryptophan indole amide peak in the in-cell spectra was used as a reference. Panels (AC) are adapted from Majumder et al. (2015) [14].
Figure 4
Figure 4
ADK quinary interaction surface does not block the active sites. (A) Relative changes in in-cell 1H-15N CRINEPT–HMQC–TROSY peak intensities of [U- 2H, 15N] ADK residues due to ribosome-mediated quinary interactions. The threshold line delineates residues whose NMR peaks undergo significant broadening. Residues that are affected by the interaction of ADK with total RNA are indicated with asterisks. (B) Residues involved in quinary interactions (red), mapped onto the molecular surface of ADK (Protein Data Bank, PDB, entry 4AKE), lie in the CORE domain of ADK. Panels (A,B) are adapted from Majumder et al. (2015) [14].
Figure 5
Figure 5
Quinary interactions of ADK in E. coli. (A) (Center) Overlay of in vitro 1H-15N CRINEPT-HMQC-TROSY spectra of 10 μM [U- 2H, 15N] ADK without (black) and with 2.5 μM ribosome (red). Surrounding panels show overlays of individual residues including in-cell NMR peaks (blue). (B) Fluorescence titration of 0.5 μM ribosome with ADK. Tryptophan fluorescence was measured at an emission wavelength of 350 nm by using an excitation wavelength of 280 nm. Curve fitting to a single site-binding isotherm yielded a Kd of 3.7 ± 0.4 μM. Fo is the fluorescence in the absence of ADK, and Fmax is the maximum fluorescence of the ADK−ribosome complex. Fluorescence titration experiments were performed in triplicate. (C) Overlay of the in-cell 1H-15N CRINEPT-HMQC-TROSY spectra of [U- 2H, 15N] ADK in the absence (blue) and presence (magenta) of 100 μg/mL chloramphenicol. K136 and A127 (left insets) in chloramphenicol treated cells exhibit chemical shift changes consistent with ATP bound ADK; G56 and S41 peaks (right insets) exhibit chemical shift changes consistent with AMP bound ADK. Panels A and B are adapted from DeMott et al. (2017) [26]. Panel (C) is adapted from Majumder et al. (2015) [14].
Figure 5
Figure 5
Quinary interactions of ADK in E. coli. (A) (Center) Overlay of in vitro 1H-15N CRINEPT-HMQC-TROSY spectra of 10 μM [U- 2H, 15N] ADK without (black) and with 2.5 μM ribosome (red). Surrounding panels show overlays of individual residues including in-cell NMR peaks (blue). (B) Fluorescence titration of 0.5 μM ribosome with ADK. Tryptophan fluorescence was measured at an emission wavelength of 350 nm by using an excitation wavelength of 280 nm. Curve fitting to a single site-binding isotherm yielded a Kd of 3.7 ± 0.4 μM. Fo is the fluorescence in the absence of ADK, and Fmax is the maximum fluorescence of the ADK−ribosome complex. Fluorescence titration experiments were performed in triplicate. (C) Overlay of the in-cell 1H-15N CRINEPT-HMQC-TROSY spectra of [U- 2H, 15N] ADK in the absence (blue) and presence (magenta) of 100 μg/mL chloramphenicol. K136 and A127 (left insets) in chloramphenicol treated cells exhibit chemical shift changes consistent with ATP bound ADK; G56 and S41 peaks (right insets) exhibit chemical shift changes consistent with AMP bound ADK. Panels A and B are adapted from DeMott et al. (2017) [26]. Panel (C) is adapted from Majumder et al. (2015) [14].
Figure 6
Figure 6
Quinary interactions of Trx in E. coli. (A) Overlay of the in-cell 1H-15N CRINEPT–HMQC–TROSY spectra of [U- 2H, 15N] Trx (blue) and that of the cellular lysate (red). The insets show overlays of the boxed regions of the in-cell spectrum (blue) and the corresponding regions of the 1H-15N CRINEPT–HMQC–TROSY spectrum of lysate (red) and the 1H-15N HSQC spectrum of purified Trx in 10 mM potassium phosphate buffer (pH 6.5) (black). The intensities of the C33, C36, I39, and G98 peaks, residues involved in quinary interactions, are broadened in-cell. (B) Overlay of the 1H-15N CRINEPT–HMQC–TROSY spectrum of [U- 2H, 15N] Trx in E. coli (blue) with crosspeaks from the 1H-15N HSQC spectrum of purified [U- 2H, 15N] Trx in 10 mM potassium phosphate buffer, pH 6.5 (black). G52, G66, and G85 exhibit broad in-cell peaks characteristic of multiple conformations of Trx in fast exchange on the NMR time scale, implying that the quinary interactions are inherently transient and dynamic. (C) Relative changes in in-cell 1H-15N CRINEPT–HMQC–TROSY crosspeak intensities of [U- 2H, 15N] Trx residues due to quinary interactions. The horizontal threshold differentiates residues whose NMR peaks undergo significant broadening. Residues annotated with asterisks are also affected in total RNA-bound Trx. (D) Residues involved in the quinary interactions (red) are mapped onto the molecular surface of Trx (PDB entry 1X0B); active site residues, C33 and G34, are in bold. The figure is adapted from Majumder et al. (2015) [14].
Figure 7
Figure 7
Binding of tetracycline and streptomycin to ribosomes changes the quinary structure of Trx in E. coli. (A) Overlay of the in-cell 1H-15N CRINEPT–HMQC–TROSY spectra of [U- 15N] Trx without (red) and with (blue) tetracycline. (B) Overlay of the in-cell 1H-15N CRINEPT–HMQC–TROSY spectra of [U- 15N] Trx without (red) and with (blue) streptomycin. Single and double asterisks indicate peaks from metabolites and unassigned side chain protons, respectively. The overlaid spectra are at the same contour levels. The reference peak used for peak intensity normalization is indicated by RP. (C) Distribution of singular values of each dataset index (binding mode) for Trx residues in the presence of tetracycline. (D) Distribution of singular values of each dataset index (binding mode) for Trx residues in the presence of streptomycin. (E) Residues involved in quinary interactions (red) due to the presence of tetracycline are mapped onto the molecular surface of Trx (Protein Data Bank entry 1X0B). (F) Residues involved in quinary interactions (red) due to the presence of streptomycin. (G) Quinary interaction surface (red) of Trx in the absence of antibiotics. Panels B–G are adapted from Breindel et al. (2017) [36].
Figure 7
Figure 7
Binding of tetracycline and streptomycin to ribosomes changes the quinary structure of Trx in E. coli. (A) Overlay of the in-cell 1H-15N CRINEPT–HMQC–TROSY spectra of [U- 15N] Trx without (red) and with (blue) tetracycline. (B) Overlay of the in-cell 1H-15N CRINEPT–HMQC–TROSY spectra of [U- 15N] Trx without (red) and with (blue) streptomycin. Single and double asterisks indicate peaks from metabolites and unassigned side chain protons, respectively. The overlaid spectra are at the same contour levels. The reference peak used for peak intensity normalization is indicated by RP. (C) Distribution of singular values of each dataset index (binding mode) for Trx residues in the presence of tetracycline. (D) Distribution of singular values of each dataset index (binding mode) for Trx residues in the presence of streptomycin. (E) Residues involved in quinary interactions (red) due to the presence of tetracycline are mapped onto the molecular surface of Trx (Protein Data Bank entry 1X0B). (F) Residues involved in quinary interactions (red) due to the presence of streptomycin. (G) Quinary interaction surface (red) of Trx in the absence of antibiotics. Panels B–G are adapted from Breindel et al. (2017) [36].
Figure 8
Figure 8
Dihydrofolate reductase, DHFR, and Thymidylate synthase, TS, engage in quinary interactions with RNA. (A) Overlay of in vitro 1H-15N CRINEPT–HMQC–TROSY spectra of 200 μM [U- 2H, 15N] DHFR with 0.5 mM folate (black) and with 0.5 mM folate and 2.5 μ M ribosome (red). Insets show individual residue overlays that include in-cell NMR peaks (blue). Folate was added to increase the solubility of DHFR. (B,C) 1H-15N HSQC spectra of 50 μΜ [U- 15N] TS with (B) 0 μg and (C) 135 μg of total E. coli RNA. The figure is adapted from DeMott et al. (2017) [26].
Figure 9
Figure 9
Ribosomes modulate ADK enzymatic activity. (A) Kinetic activity profile for ADK without (black) and with (red) 1 μM ribosome. (B) Overlays of in vitro 1H-15N CRINEPT–HMQC–TROSY spectra of 10 μM [U- 2H, 15N] ADK at 0 μM adenosine triphosphate, ATP, (blue), 20 μM ATP (magenta), 40 μM ATP (black), and 80 μM ATP plus 1 μM ribosome (red). (C) ATP analogue β,γ-methyleneadenosine 5′-triphosphate, AMP-PCP binding to ribosomes. The concentration of ribosomes was 2 μM. The figure is adapted from DeMott et al. (2017) [26].
Figure 10
Figure 10
Ribosomes modulate TS and DHFR enzymatic activities. (A) Function linkage between TS and DHFR in the thymidylate synthetic pathway (B) Increase in TS activity with increasing ribosome concentration. (C) Kinetic activity profile for TS without (black) and with (red) 0.5 μM ribosome. (D) Kinetic activity profile for DHFR without (black) and with (red) 0.5 μM ribosome. (E) NADPH binding to ribosomes. The concentration of ribosomes was 1 μM. Figure is adapted from DeMott et al. (2017) [26].

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