Large-Scale Isolation of Proteins of the Large Subunit fromEscherichia coliRibosomes

Diedrich, Gundo; Burkhardt, Nils; Nierhaus, Knud H.

doi:10.1006/prep.1996.0702

Cited by 13 publications

(8 citation statements)

References 21 publications

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“…1B, some residual secondary or tertiary structure impedes the reaction of SMTA at one site in the protein, resulting in less than total modification. In past results using the ribosomal proteins from organisms with less extreme optimal growth temperatures, 20–37°C, it has been typical to observe some evidence of less than complete SMTA derivatization with proteins such as L7/L12 that are noted for retaining significant structure even under strongly denaturing conditions such as our SCX separation conditions 60.…”

Section: Discussionmentioning

confidence: 83%

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

Running

Reilly

2010

Proteomics

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Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.

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Section: Discussionmentioning

confidence: 83%

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

Running

Reilly

2010

Proteomics

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“…Ribosomal protein L7/L12 is known to retain some structure and to self-associate even under strongly denaturing conditions, such as the 6 M urea solutions used by ourselves and others for SCX chromatography. 53,54 The robust structure of this protein explains its partial reactivity, and a very stable structure is also the most likely explanation for incomplete modification of S11 in the disassembled, denatured reactions. Examples of the completeness of labeling of other proteins are shown in Figures 1C, 2C, 3C and Supplemental Figure 2C (Supporting Information).…”

Section: Resultsmentioning

confidence: 99%

Ribosomal Proteins of Deinococcus radiodurans: Their Solvent Accessibility and Reactivity

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Reilly

2009

J. Proteome Res.

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The structure of proteins in native ribosomes from Deinococcus radiodurans R1 was probed by S-methylthioacetimidate (SMTA) modification of amino groups. The extent of protein labeling was quantified using top down methods, and modified positions were identified using bottom up experiments. Each protein's reactivity was predicted by examination of the crystal structures of the D. radiodurans 50S subunit and the T. thermophilus HB8 30S subunit. The close phylogenetic relation between D. radiodurans and T. thermophilus allowed the evaluation of D. radiodurans small subunit protein reactivity by alignment of homologous sequences. As a result, we were able to observe and characterize the reactivity of all of D. radiodurans ribosomal proteins. The extent of protein amidination was well correlated with the solvent-exposed surface area of each protein and even better correlated with the number of visible lysine residues. Lysine residues that are in close contact with rRNA structural features or buried in protein tertiary structure are nonreactive with SMTA, while those that are surface exposed are modified. Crystallographic disorder and post-translational modifications lead to differences between the observed and predicted extents of reactivity. Comparison of unmodified and disassembled amidinated protein mixtures also shows great promise for the quality control of the proteomic sequences and has facilitated the identification of four sequencing errors in the ribosomal proteome of D. radiodurans R1.

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“…Similar interactions and coelution of proteins from E. coli ribosomes were observed by Diederich in a similar ionexchange chromatography system. 73 Band broadening in whole protein chromatography due to heterogeneous protein binding is a well-recognized problem, and there are three commonly applied tactics to minimize the effects: increasing flow rates, increasing temperature, and the addition of organic solvents to the mobile phase. [75][76][77][78] The modular nature of our system would allow the addition of a thermocouple-controlled heater to the ion-exchange column.…”

Section: Discussionmentioning

confidence: 99%

A Top-Down/Bottom-Up Study of the Ribosomal Proteins of Caulobacter crescentus

et al. 2006

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Ribosomes from the gram-negative α-proteobacterium Caulobacter crescentus were isolated using standard methods. Proteins were separated using a two-dimensional liquid chromatographic system that allowed the analysis of whole proteins by direct coupling to an ESI-QTOF mass spectrometer and of proteolytic digests by a number of mass spectrometric methods. The masses of 53 of 54 ribosomal proteins were directly measured. Protein identifications and proposed post-translational modifications were supported by proteolysis with trypsin, endoprotease Glu-C and exoproteases carboxypeptidases Y and P. Tryptic peptide mass maps show an average sequence coverage of 62%, and carboxypeptidase C-terminal sequence tagging provided unambiguous identification of the small, highly basic proteins of the large subunit. C. crescentus presents some post-translational modifications that are similar to those of E. coli (e. g. N-terminal acetylation of S9 and S18) along with some unique variations, such as a near absence of L7 and extensive modification of L11. The comprehensive description of this organism's ribosomal proteome provides a foundation for the study of ribosome structure, dependence of post-translational modifications on growth conditions, and the evolution of subcellular organelles.

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Large-Scale Isolation of Proteins of the Large Subunit fromEscherichia coliRibosomes

Cited by 13 publications

References 21 publications

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

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

Ribosomal Proteins of Deinococcus radiodurans: Their Solvent Accessibility and Reactivity

A Top-Down/Bottom-Up Study of the Ribosomal Proteins of Caulobacter crescentus

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