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The solution structure of ribosomal protein L18 from Thermus thermophilus reveals a conserved RNA-binding fold
KTH, Superseded Departments, Biotechnology.
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2002 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 363, no 3, 553-561 p.Article in journal (Refereed) Published
Abstract [en]

We have determined the solution structure of ribosomal protein L18 from Thermus thermophilus. L18 is a 12.5 kDa protein of the large subunit of the ribosome and binds to both 5 S and 23 S rRNA. In the uncomplexed state L18 folds to a mixed α/β globular structure with a long disordered N-terminal region. We compared our high-resolution structure with RNA-complexed L 18 from Haloarcula marismortui and T. thermophilus to examine RNA-induced as well as species-dependent structural differences. We also identified T. thermophilus S11 as a structural homologue and found that the structures of the RNA-recognition sites are conserved. Important features, for instance a bulge in the RNA-contacting β-sheet, are conserved in both proteins. We suggest that the L18 fold recognizes a specific RNA motif and that the resulting RNA-protein-recognition module is tolerant to variations in sequence.

Place, publisher, year, edition, pages
2002. Vol. 363, no 3, 553-561 p.
Keyword [en]
NMR spectroscopy, Protein structure, Ribosome, RNA-binding protein
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-5332DOI: 10.1042/0264-6021:3630553ISI: 000175651600015PubMedID: 11964156OAI: oai:DiVA.org:kth-5332DiVA: diva2:8633
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Protein production, characterization and structure determination in structural genomics
Open this publication in new window or tab >>Protein production, characterization and structure determination in structural genomics
2004 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis covers the process from expression of a heterologous gene in Escherichia coli to structure determination of a protein by nuclear magnetic resonance (NMR) spectroscopy.

The first part concerns structural genomics-related parallel screening studies on the effect of fusion tags (in particular the His tag) on protein solubility and the use of fusion tags in fast, parallel purification protocols intended for initial biophysical characterization of human proteins produced in E. coli. It was found that for most proteins the His tag has a negative influence on protein solubility. This influence appears to be more pronounced for our C-terminal His tag than for the N-terminal His tags used in this study. Moreover, high ratios of soluble per total protein do not always guarantee a high yield of soluble protein after purification, as different vector - target protein combinations result in large differences in host cell growth rates. Protein purification protocols for different fusion tags were developed that make it possible to express, purify and study structural properties of low concentration samples of 15N-labeled proteins in one or two days.

The second part of this thesis describes the assignment and solution structure determination of ribosomal protein L18 of Thermus thermophilus. The protein is a mixed α/β structure with two α-helices on one side of a four-stranded β-sheet. Comparison to RNA-bound L18 showed that the protein to a large extent adopts identical structures in free and bound states, with exception of the loop regions and the flexible N-terminus.

Keywords: protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, ribosomal protein

Place, publisher, year, edition, pages
Bioteknologi, 2004
Keyword
Biology, protein production, protein solubility, fusion tags, nuclear magnetic resonance, structure determination, Biologi
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-29 (URN)91-7283-838-8 (ISBN)
Public defence
2004-10-01, FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-10-01 Created: 2004-10-01 Last updated: 2012-03-22

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