Secondary structural analysis of retrovirus integrase: Characterization by circular dichroism and empirical prediction methods

Lin, Thy‐Hou; Quinn, Thomas P.; Grandgenett, Duane P.; Walsh, Mary T.

doi:10.1002/prot.340050210

Cited by 22 publications

(8 citation statements)

References 53 publications

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“…These discrepancies were consistent with the validity of the prediction methods estimated at 60 % (Biou et al, 1988). The same discrepancies have been observed in different proteins in work by other authors (Lin et al, 1989). Furthermore Busetta & Hospital (1982) have shown that proteins with a mixed-type (a//3) structure have worse prediction levels than those with a single type of secondary structure (all a-helix or all /3-structure).…”

Section: Discussionsupporting

confidence: 85%

“…The helix content was also estimated from the Ae value at 222 nm by the equation described by Lin et al (1989). The amount of a-helix calculated for the factor VIII light chain was 220%, which is consistent with the result obtained using the curve-fitting procedure.…”

Section: Vol 288supporting

confidence: 83%

“…The present experimental results indicated that human factor VIII light chain contains 42 % irregular structure, 36 % /strands, and 22 % a-helix. Using the equation reported by Lin et al (1989), we have obtained the same helical content directly from the c.d. spectrum.…”

Section: Discussionmentioning

confidence: 99%

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First determination of the secondary structure of purified factor VIII light chain

Bihoreau

Fontaine‐Aupart

Lehegarat

et al. 1992

Biochemical Journal

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The first analysis of the secondary structure of human factor VIII light chain was performed by c.d. spectroscopy. The purification process described in this paper allowed us to obtain the large amounts of purified factor VIII light chains required for c.d. experiments. Since this 80 kDa protein is non-covalently associated with a heavy chain to form the active molecule, isolated factor VIII light chains were obtained after immunoadsorption and dissociation of the immobilized active complexes by EDTA. Furthermore, factor VIII light chains were discriminated from the residual active complexes and the free heavy chains by a final ion-exchange-chromatography step. This f.p.l.c. analysis showed that factor VIII light chains were less electronegative than the active complexes. The results of conformational analysis by c.d. show that the protein possesses a high degree of regular secondary structure (58%) with approx. 22% of alpha-helix and 36% of beta-strand structures. The protein was completely unfolded by 3 M-guanidine hydrochloride. The results obtained from the analysis of c.d. spectra were compared with those predicted from three different statistical methods based on amino-acid sequence. The secondary structure information obtained from these methods was in good agreement with the c.d. results. These results were comparable with the secondary structure prediction of ceruloplasmin, a protein known to show sequence identity to factor VIII.

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Section: Discussionsupporting

confidence: 85%

Section: Vol 288supporting

confidence: 83%

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First determination of the secondary structure of purified factor VIII light chain

Bihoreau

Fontaine‐Aupart

Lehegarat

et al. 1992

Biochemical Journal

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“…IN proteins from different retroviral species vary in size and amino acid homology (20,25,29). Two regions of strong similarity are shared between retroviral IN proteins, a proposed zinc finger motif or HHCC region in the amino terminus, and a central core region containing the DD (35)E motif.…”

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confidence: 99%

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis

Jonsson¹,

Donzella²,

Gaucan³

et al. 1996

J Virol

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Retroviral integrases perform two catalytic steps, 3 processing and strand transfer, that result in the stable insertion of the retroviral DNA into the host genome. Mutant M-MuLV integrases were constructed to define the functional domains important for 3 processing, strand transfer, and disintegration by in vitro assays. N-terminal mutants had no detectable 3 processing activity, and only one mutant which lacks the HHCC domain, N⌬105, had strand transfer activity. Strand transfer mediated by N⌬105 showed preference for one site in the target DNA. Disintegration activity of N-terminal mutants decreased only minimally. In contrast, all C-terminal mutants truncated by more than 28 amino acids had no integration or disintegration activity. Activity on a single-strand disintegration substrate did not require a functional HHCC domain but did require most of the C-terminal region. Complementation analysis found that the HHCC region alone was able to function in trans to a promoter containing only the DD(35)E and C-terminal regions and to enhance integration site selection. Increasing the reducing conditions or adding the HHCC domain to N⌬105 reaction mixtures restored the wild-type strand transfer activity and range of target sites. The reducing agent affected Cys-209 in the DD(35)E region. The presence of C-209 was required for complementation of N⌬105 by the HHCC region.

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“…The negative band at 222 nm indicates the onset of the formation of α-helical conformations. In a first approximation, we calculated a helix content of 22%, and 11% for the samples with least PEG-DGE and BS3 respectively [22][23][24]. For high degrees of crosslinking, the CD spectra indicated a fully disordered elastin structure.…”

Section: Resultsmentioning

confidence: 99%

Stimuli-Sensitive Microgels from Native Elastin: An Easy Approach for a Drug Release System

Singh

Topuz

Albrecht

et al. 2013

Hierarchical Macromolecular Structures: 60 Years After the Staudinger Nobel Prize II

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Thermo-and pH-responsive microgels were prepared from solubilized native elastin by crosslinking of the elastin lysine residues with poly(ethylene glycol) diglycidyl ether (PEG-DGE) and with bis(sulfosuccinimidyl) suberate (BS3). In the first case, a peptide-PEG conetwork was obtained whereas, in the second case, the elastin peptides were interlinked with hydrophobic bridges. The structure of the microgels was controlled by the ratio of crosslinker to elastin and by performing the crosslinking reaction in an inverse minielemulsion, yielding particles with a diameter in the submicron range. Depending on the degree of crosslinking, the hybrid microgels exhibited a volume change transition at 37 and 35.5 C and pH responsivity in the range of 5-7 for microgels crosslinked with PEG-DE and BS3, respectively. This temperature-and pH-responsive behavior can be assigned to the well-investigated coacervation of elastin peptides, demonstrating that the elastin functionality is abolished only by rather dense crosslinking. In spite of the broad distribution in the molecular weight of the elastin molecules, the microgels remained soluble. Light scattering and sedimentation experiments demonstrated that the coacervation occurred predominantly intramolecularly, i.e., by collapse in the core while the corona stabilized the colloidal dispersion against precipitation. Preliminary experiments were conducted to evaluate the suitability of these microgels for use as a drug-release system and demonstrated

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Secondary structural analysis of retrovirus integrase: Characterization by circular dichroism and empirical prediction methods

Cited by 22 publications

References 53 publications

First determination of the secondary structure of purified factor VIII light chain

First determination of the secondary structure of purified factor VIII light chain

Functional domains of Moloney murine leukemia virus integrase defined by mutation and complementation analysis

Stimuli-Sensitive Microgels from Native Elastin: An Easy Approach for a Drug Release System

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