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

David Burz,Leonard Breindel,Alexander Shekhtman

doi:10.3390/ijms20061297

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.

Highlights

For the past two decades, in-cell NMR spectroscopy has been used to investigate the structure, dynamics and interaction surfaces of proteins inside living cells [1,2,3,4,5,6,7]
The absence of widespread line broadening in early experiments performed in E. coli was due to the fact that the overexpressed proteins leaked out of the cells during in-cell NMR experiments [43] or overexpression of labeled target exceeded 100 μM [13], which is ≥10 times greater than the estimated dissociation constant of 1–10 μM for target protein quinary interactions
The 1H-15N cross relaxation-induced polarization transfer (CRINEPT)–heteronuclear multiple quantum coherence (HMQC)–TROSY NMR spectrum of 10 μM [U- 2H, 15N] Adenylate kinase (ADK) collected in vitro in the presence of 2.5 μM ribosomes exhibited broadened peaks that largely coincide with the in-cell spectrum (Figure 2D)

Summary

Introduction

For the past two decades, in-cell NMR spectroscopy has been used to investigate the structure, dynamics and interaction surfaces of proteins inside living cells [1,2,3,4,5,6,7]. Over the past few years, work in our laboratory has suggested that RNA, in particular ribosomes, plays a major role in establishing protein quinary structures [14,26] This conclusion is in general agreement with mass spectroscopic studies of mRNA- and ribo-interactomes [27,28,29,30] in which hundreds of eukaryotic proteins bound to either mRNA or ribosomes were identified and did not possess obvious RNA binding motifs. Such observations have provided a glimpse of insight into the physical complexity of quinary interactions [31,32,33,34]. Effect of RNA-Binding Blocks polyubiquitination sites, increases apparent MW [14] Increases apparent MW [14] Antibiotic binding to ribosome alters quinary structure [36] Increases apparent MW [14] Noncompetitive kinetic inhibitor [26]

Protein-RNA Interactions Broaden Target Protein NMR Spectra

Resolving Target Protein Bound to RNA

Adenylate Kinase

Dihydrofolate Reductase and Thymidylate Synthase

Findings

Discussion