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Charlotte Rohde Knudsen

Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.

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Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach. / Laurberg, M; Mansilla, Francisco; Clark, Brian F. C.; Knudsen, Charlotte Rohde.

I: Journal of Biological Chemistry, Bind 273, Nr. 8, 1998, s. 4387-91.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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Laurberg, M, Mansilla, F, Clark, BFC & Knudsen, CR 1998, 'Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.' Journal of Biological Chemistry, bind 273, nr. 8, s. 4387-91.

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Author

Laurberg, M ; Mansilla, Francisco ; Clark, Brian F. C. ; Knudsen, Charlotte Rohde. / Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach. I: Journal of Biological Chemistry. 1998 ; Bind 273, Nr. 8. s. 4387-91.

Bibtex

@article{5e91c690da4611dcbc43000ea68e967b,
title = "Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.",
abstract = "The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region. Udgivelsesdato: 1998-Feb-20",
keywords = "Escherichia coli, GTP Phosphohydrolase-Linked Elongation Factors, Guanosine Diphosphate, Guanosine Triphosphate, Hydrolysis, Mutagenesis, Site-Directed, Peptide Elongation Factor Tu, Protein Engineering, Recombinant Proteins",
author = "M Laurberg and Francisco Mansilla and Clark, {Brian F. C.} and Knudsen, {Charlotte Rohde}",
year = "1998",
language = "English",
volume = "273",
pages = "4387--91",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "8",

}

RIS

TY - JOUR

T1 - Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach.

AU - Laurberg, M

AU - Mansilla, Francisco

AU - Clark, Brian F. C.

AU - Knudsen, Charlotte Rohde

PY - 1998

Y1 - 1998

N2 - The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region. Udgivelsesdato: 1998-Feb-20

AB - The function of the N-terminal region of elongation factor Tu is still unexplained. Until recently, it has not been visible in electron density maps from x-ray crystallography studies, but the presence of several well conserved basic residues suggest that this part of the molecule is of structural importance for the factor to function properly. In this study, two lysines at positions 4 and 9 were mutated separately to alanine or glutamate. The resulting four point mutants were expressed and purified using the pGEX system. The untagged products were characterized with regard to guanine-nucleotide interaction, intrinsic GTPase activity, and binding of aminoacyl-tRNA (aa-tRNA). The results show that Lys9 is especially strongly involved in the association with guanine nucleotides and the binding of aa-tRNA. Also Lys4 plays a role in the association of GDP and GTP and is also of some importance in aa-tRNA binding. Our results are discussed in structural terms with the conclusion that a complex network of interactions across the interface between domains 1 and 2 with Lys9 being a key residue seems to be important for the fine tuning of the dimensions of the cleft accommodating the acceptor end of aa-tRNA as well as delineating the structure of the effector region. Udgivelsesdato: 1998-Feb-20

KW - Escherichia coli

KW - GTP Phosphohydrolase-Linked Elongation Factors

KW - Guanosine Diphosphate

KW - Guanosine Triphosphate

KW - Hydrolysis

KW - Mutagenesis, Site-Directed

KW - Peptide Elongation Factor Tu

KW - Protein Engineering

KW - Recombinant Proteins

M3 - Journal article

VL - 273

SP - 4387

EP - 4391

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 8

ER -