Limited proteolysis analysis of the ribosome is affected by subunit association

Abstract

Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from mis-folding leading to their eventual degradation within the cell, is limited due to the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X-ray crystallography and cryo-EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits. Isolated subunits were analyzed using limited proteolysis and MALDI-MS and the results were compared to previous data obtained from 70S ribosomes. Generally, ribosomal proteins were found to be more stable in 70S ribosomes than in their isolated subunits, consistent with a reduction in conformational flexibility upon subunit assembly. This approach demonstrates that limited proteolysis combined with MALDI-MS can reveal structural changes to ribosomes upon subunit assembly or disassembly, and provides the appropriate benchmark data from 30S, 50S and 70S proteins to enable studies of ribosome assembly intermediates.

Molecular Determinants of Enzyme Cold Adaptation: Comparative Structural and Computational Studies of Cold- and Warm-Adapted Enzymes

“…Techniques that provide a relative estimation of structural flexibility exist, but they in several cases do not directly measure flexibility, but some properties correlated to it, as relative resistance to proteolysis [94][95][96], fluorescence of tryptophan or tyrosine residues [97][98][99][100], hydrogen/deuterium (H/D) exchange experiments monitored by NMR [101,102], Fourier Transform Infrared Spectroscopy (FT-IR) [103,104] or ElectroSpray Ionization-Mass Spectrometry (ESI-MS) [105,106], experiments with fluorescent ANS dye , Electron Paramagnetic Resonance (EPR) [108][109][110], Single Molecule Foster Resonance Energy Transfer (FRET) [111]. Therefore, these techniques are often unable to identify specific but relevant regions of localized flexibility, because their estimations describe, in many cases and with few exceptions, overall properties.…”

Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH

“…These include hydrogen–deuterium exchange 10–12, and covalent modification 13–23. Covalent modification techniques can either decrease the masses of proteins, as is seen with limited proteolysis of native structures to remove conformationally flexible “loops and fringes” of a protein 13–16, or increase protein masses, as in selective modification of specific amino acid side chains 17–24. In contrast to hydrogen–deuterium exchange, covalent modifications are usually not labile, which simplifies the handling and analysis of the labeled proteins.…”

A systematic study of 50S ribosomal subunit purification enabling robust crystallization

“…This portion is absent in the D. radiodurans crystal structure, while the homologous sequence in the T. thermophilus 70S crystal structure extends towards the 30S subunit in a helical conformation. Protein L19 is involved in intersubunit contacts in the 70S ribosome and has been found to be protected from tryptic digestion by subunit association (Hamburg et al, 2009).…”