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  Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process

Divita, G., Rittinger, K., Geourjon, C., Deléage, G., & Goody, R. S. (1995). Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process. Journal of Molecular Biology (London), 245(5), 508-521. doi:10.1006/jmbi.1994.0042.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-48DC-9 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-48DD-7
Genre: Journal Article
Alternative Title : Dimerization kinetics of HIV-1 and HIV-2 reverse transcriptase: a two step process

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JMolBiol_245_1995_508.pdf (Any fulltext), 898KB
 
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 Creators:
Divita, Gilles1, Author              
Rittinger, Katrin1, Author              
Geourjon, Christophe, Author
Deléage, Gilbert, Author
Goody, Roger S.1, Author              
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1Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society, ou_1497712              

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Free keywords: Reverse transcriptase; protein folding; fluorescence; dimerization kinetic; HIV
 Abstract: The dimerization processes of the human immunodeficiency virus (HIV) types 1 and 2 reverse transcriptase (RTs) from their subunits have been investigated using a number of complementary approaches (fluorescence spectroscopy, size exclusion-HPLC and polymerase activity assay). The formation of the native heterodimeric form of HIV-1 and HIV-2 RT occurs in a two step process. The first step is a concentration-dependent association of the two subunits (p66 and p51) to give a heterodimeric intermediate, which slowly isomerizes to the "mature" heterodimeric form of the enzyme. For both RTs, the first step behaves as a second order reaction with similar association rate constants (in the range of 2 x 10(4) to 4 x 10(4) M-1 s-1). This initial dimerization results in a 25% quenching of the intrinsic fluorescence and a 30% decrease in the accessibility of the tryptophan hydrophobic cluster to solvent as revealed by iodide quenching experiments and by monitoring the binding of 1-anilino-8-naphthalenesulphonate. The formation of the intermediate-RT form appears to involve hydrophobic regions of the subunits containing tryptophan residues. This intermediate form is devoid of polymerase activity, but is able to bind primer/template with high affinity. The final stage of the mature RT-heterodimer formation occurs in a slow first order reaction, which is 12-fold faster for HIV-2 (1.2 h-1) than HIV-1 RT (0.1 h-1). At micromolar concentrations, this slow isomerization constitutes the rate limiting step of the RT maturation and the structural change involved appears to be partly associated with the catalytic site, as shown using fluorescent labelled primer/template. On the basis of both the presently available X-ray structure of the HIV-1 RT and the predicted structure of HIV-2 RT, the thumb subdomain of the p51 subunit seems to be involved in this maturation step, which is probably the interaction of this domain with the RNAse H domain of the large subunit. The placement of the fingers subdomain of p51 in the palm subdomain of the p66 subunit may also be associated with formation of mature heterodimeric RTs.

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Language(s): eng - English
 Dates: 1994-08-191994-10-251995-02-03
 Publication Status: Published in print
 Pages: 14
 Publishing info: -
 Table of Contents: -
 Rev. Method: Peer
 Degree: -

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Title: Journal of Molecular Biology (London)
  Other : J Mol Biol
Source Genre: Journal
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Publ. Info: London : Academic Press
Pages: - Volume / Issue: 245 (5) Sequence Number: - Start / End Page: 508 - 521 Identifier: ISSN: 0022-2836
CoNE: /journals/resource/954922646042