The tertiary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by proton magnetic resonance spectroscopy and limited proteolysis experiments

Littlechild, J.A.; Malcolm, Ailsa; Paterakis, Kostas; Ackermann, Isolde; Dijk, Jan

doi:10.1016/0167-4838(87)90336-0

Cited by 15 publications

(6 citation statements)

References 32 publications

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“…As seen in Table 1, the proteolytic fragments generally arise from cleavage of N-and/or C-terminal domains of the various r-proteins. Similar cleavages have been found during limited proteolysis studies of isolated r-proteins whose tertiary structure was maintained after extraction from ribosomes [40]. These data demonstrate that direct MALDI-MS analysis of limited proteolysis mixtures is compatible with both the identification of proteins susceptible to enzymatic proteolysis as well as defining the sequence location of proteolysis.…”

supporting

confidence: 77%

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Suh

Pourshahian

Limbach

2007

J. Am. Soc. Mass Spectrom.

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An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30-60 min of incubation provide insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.The ribosome, found in all organisms, is the subcellular organelle that performs the activity of protein synthesis. Prokaryotic ribosomes, which sediment at 70S, have a molecular mass of approximately 2.5 ×10 6 Da and consist of two subunits, the 50S and the 30S. Each subunit has a unique number of proteins and ribosomal RNAs (rRNAs). Eukaryotic ribosomes, which sediment at 80S, are substantially larger and more complex than their prokaryotic counterparts. Two subunits, the 60S and 40S, together contain at least 78 unique proteins and 4 rRNAs. The small subunit binds messenger RNA (mRNA) and mediates the interactions between mRNA and transfer RNAs (tRNAs). The larger subunit catalyzes peptide-bond formation. During the initiation phase of protein synthesis, the two subunits behave independently, assembling into complete ribosomes only when elongation is about to begin.A fundamental prerequisite for understanding the biochemical interactions occurring within the ribosome during protein synthesis is a detailed knowledge of the structure of this † Current address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10021 * To whom correspondence should be addressed. Phone (513) 556-1871, Fax (513) Email: Pat.Limbach@uc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

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supporting

confidence: 77%

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Suh

Pourshahian

Limbach

2007

J. Am. Soc. Mass Spectrom.

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“…Nonetheless, CD spectroscopy is quite useful for comparing the degrees of structure in different preparations of related proteins, as was done here. The proton NMR spectra for the proteins measured by Littlechild and collaborators (23) and Morrison et al (26) were also similar to ours, although they concluded that this technique could not entirely discount the presence of structure. However, given the similarity of the CD and 1D proton NMR spectra, together with the 2D NMR spectrum, we conclude that E. coli L27 is unstructured in solution regardless of the method of isolation.…”

Section: Vol 183 2001supporting

confidence: 66%

Differential Effects of ReplacingEscherichiacoliRibosomal Protein L27 with Its Homologue fromAquifexaeolicus

Maguire¹,

Manuilov

Zimmermann

2001

J Bacteriol

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The rpmA gene, which encodes 50S ribosomal subunit protein L27, was cloned from the extreme thermophile Aquifex aeolicus, and the protein was overexpressed and purified. Comparison of the A. aeolicus protein with its homologue from Escherichia coli by circular dichroism analysis and proton nuclear magnetic resonance spectroscopy showed that it readily adopts some structure in solution that is very stable, whereas the E. coli protein is unstructured under the same conditions. A mutant of E. coli that lacks L27 was found earlier to be impaired in the assembly and function of the 50S subunit; both defects could be corrected by expression of E. coli L27 from an extrachromosomal copy of the rpmA gene. When A. aeolicus L27 was expressed in the same mutant, an increase in the growth rate occurred and the "foreign" L27 protein was incorporated into E. coli ribosomes. However, the presence of A. aeolicus L27 did not promote 50S subunit assembly. Thus, while the A. aeolicus protein can apparently replace its E. coli homologue functionally in completed ribosomes, it does not assist in the assembly of E. coli ribosomes that otherwise lack L27. Possible explanations for this paradoxical behavior are discussed.

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“…As well as providing a means to probe the interactions between the components of the ribosome, the observation of distinct peaks in the mass spectrometer provides an opportunity to investigate the properties of individual proteins. Many of these are at least partially unfolded in isolation (26), and little is known about their structure and dynamics in the intact particle. A powerful means of probing the latter is through hydrogen͞deuterium (H͞D) exchange experiments (27), which can be monitored by mass spectrometry as a consequence of the resulting changes in mass (28 -31).…”

Section: Fig1mentioning

confidence: 99%

Mass spectrometry of ribosomes and ribosomal subunits

Benjamin

Robinson

Hendrick

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

103

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Nanof low electrospray ionization has been used to introduce intact Escherichia coli ribosomes into the ion source of a mass spectrometer. Mass spectra of remarkable quality result from a partial, but selective, dissociation of the particles within the mass spectrometer. Peaks in the spectra have been assigned to individual ribosomal proteins and to noncovalent complexes of up to five component proteins. The pattern of dissociation correlates strongly with predicted features of ribosomal protein-protein and protein-RNA interactions. The spectra allow the dynamics and state of folding of specific proteins to be investigated in the context of the intact ribosome. This study demonstrates a potentially general strategy to probe interactions within complex biological assemblies.Mass spectrometry is well established as a powerful and rapidly expanding technique for studying the covalent structures of biological macromolecules. The development of electrospray ionization (ESI) has extended the reach of mass spectrometry into the realm of noncovalent interactions (1). ESI techniques can generate gas-phase ions from solutions in which proteins are in their native state, allowing investigation of their structure and dynamics and events associated with their folding (2). Combined with the sensitivity and generous molecular weight limits of modern mass analyzers, ESI holds the promise of allowing studies of noncovalent structure in large multimolecular complexes (3, 4). Here we report on the successful use of ESI mass spectrometry to initiate investigations of ribosomes from Escherichia coli.The pivotal role of the ribosome in the translation of genetic information into biological activity has made it the subject of intensive research over the past 30 years, revealing much about its structure and function (5-7). Most of this research has been directed toward E. coli ribosomes. These 2.5-MDa particles contain 54 proteins, many of which are posttranslationally modified, and three large RNA molecules (also posttranscriptionally modified), which comprise the majority of the mass of the ribosome. The largest RNA molecule is thought to be a ribozyme participating in the peptidyltransferase reaction (8, 9). Intact (70S) ribosomes consist of two noncovalently associated subunits: a small (30S) subunit and a large (50S) subunit, each of which is stable in isolation. Interactions within and between the subunits determine and regulate the process of protein biosynthesis within the cell. MATERIALS AND METHODSRibosomes. Ribosomes were harvested from E. coli strain MRE600 by using standard protocols (10); no salt washing steps were used. Stock solutions then were buffer-exchanged to a final concentration of 50-500 nM in 10 mM ammonium chloride, 1 mM 2-mercaptoethanol, pH 6.5 by using Centricon 100 concentrators. Any monomeric proteins that dissociated from the ribosome during this exchange protocol passed through the membrane. Denatured ribosomes were prepared by suspension of stock ribosomes in 50% acetic acid at 0°C for 30 min (...

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The tertiary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by proton magnetic resonance spectroscopy and limited proteolysis experiments

Cited by 15 publications

References 32 publications

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Differential Effects of ReplacingEscherichiacoliRibosomal Protein L27 with Its Homologue fromAquifexaeolicus

Mass spectrometry of ribosomes and ribosomal subunits

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