Imperial College London
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ProfessorIanGould

Faculty of Natural Sciences, Department of Chemistry

Senior Research Investigator
 
 
 
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Contact

 

+44 (0)20 7594 5809i.gould

 
 
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Location

 

110BMolecular Sciences Research HubWhite City Campus

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Summary

 

Publications

Citation

BibTex format

@article{Hughes:2003:10.1186/1472-6807-3-5,
author = {Hughes, SJ and Tanner, JA and Hindley, AD and Miller, AD and Gould, IR},
doi = {10.1186/1472-6807-3-5},
journal = {BMC Struct Biol},
title = {Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment.},
url = {http://dx.doi.org/10.1186/1472-6807-3-5},
volume = {3},
year = {2003}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - BACKGROUND: Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. RESULTS: Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. CONCLUSIONS: The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-t
AU  - Hughes,SJ
AU  - Tanner,JA
AU  - Hindley,AD
AU  - Miller,AD
AU  - Gould,IR
DO  - 10.1186/1472-6807-3-5
PY  - 2003///
TI  - Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment.
T2  - BMC Struct Biol
UR  - http://dx.doi.org/10.1186/1472-6807-3-5
UR  - https://www.ncbi.nlm.nih.gov/pubmed/12787471
VL  - 3
ER  -
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