Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network

Andrade, Miguel Ángel Gutiérrez; Chacón, Pablo; Merelo, J. J.; Morán, Federico

doi:10.1093/protein/6.4.383

Cited by 992 publications

(833 citation statements)

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“…Hsp33 ox43°C has substantially less α-helical content than Hsp33 red . Secondary structure predictions by K2D 17 show a large decrease in the α-helical content, from 37% in Hsp33 red to 19% in Hsp33 ox43°C (Supplementary Table 1 online). This unfolding causes either the formation or exposure of hydrophobic surfaces that are probably the substrate-binding site(s) in Hsp33 (Fig.…”

Section: Identification Of a Thermolabile Region In Hsp33 Ox30°cmentioning

confidence: 99%

The redox-switch domain of Hsp33 functions as dual stress sensor

Ilbert

Horst

Ahrens

et al. 2007

Nat Struct Mol Biol

170

207

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The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.Reactive oxygen species develop as unavoidable consequences of aerobic life. Their oxidizing effects on most cellular macromolecules can be deleterious to cells and organisms 1 . Cells have developed effective antioxidant systems to counteract this hazard (for review, see ref. 2 ). Disruption of the fine balance between oxidants and antioxidants, however, leads to the accumulation of reactive oxygen species and to a condition termed oxidative stress 3 . Oxidative stress conditions have been shown to develop in, and may even cause, numerous physiological and pathological conditions, such as aging, heart disease, diabetes and neurodegenerative diseases 4 .

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Section: Identification Of a Thermolabile Region In Hsp33 Ox30°cmentioning

confidence: 99%

The redox-switch domain of Hsp33 functions as dual stress sensor

Ilbert

Horst

Ahrens

et al. 2007

Nat Struct Mol Biol

170

207

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“…To analyze the CD spectrum the following algorithms were used: VARSLC, 42 SELCON3, 43 CONTINLL, 44 and K2D. 45 All methods as incorporated into the program Dicroprot 46 and NeuralNet. 47 …”

Section: Circular Dichroism Spectroscopymentioning

confidence: 99%

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

et al. 2010

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V-ATPase is a multisubunit membrane complex that functions as nanomotor coupling ATP hydrolysis with proton translocation across biological membranes. Recently, we uncovered details of the mechanism of interaction between the N-terminal tail of the V-ATPase a2-subunit isoform (a2N ) and ARNO, a GTP/GDP exchange factor for Arf-family small GTPases. Here, we describe the development of two methods for preparation of the a2N 1-402 recombinant protein in milligram quantities sufficient for further biochemical, biophysical, and structural studies. We found two alternative amphiphilic chemicals that were required for protein stability and solubility during purification: (i) non-detergent sulfobetaine NDSB-256 and (ii) zwitterionic detergent FOS-CHOLINEV R 12 (FC-12). Moreover, the other factors including mild alkaline pH, the presence of reducing agents and the absence of salt were beneficial for stabilization and solubilization of the protein. A preparation of a2N 1-402 in NDSB-256 was successfully used in pull-down and BIAcore TM protein-protein interaction experiments with ARNO, whereas the purity and quality of the second preparation in FC-12 was validated by size-exclusion chromatography and CD spectroscopy. Surprisingly, the detergent requirement for stabilization and solubilization of a2N 1-402 and its cosedimentation with liposomes were different from peripheral domains of other transmembrane proteins. Thus, our data suggest that in contrast to current models, so called ''cytosolic'' tail of the a2-subunit might actually be embedded into and/or closely associated with membrane phospholipids even in the absence of any obvious predicted transmembrane segments. We propose that a2N 1-402 should be categorized as an integral monotopic domain of the a2-subunit isoform of the V-ATPase.

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“…Data were scaled to molar ellipticity (in deg cm 2 dmol À1 ). Secondary structure content was estimated using K2D [24].…”

Section: Circular Dichroism Spectropolarimetrymentioning

confidence: 99%

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

Stanley

Versluis

Schultz

et al. 2007

Archives of Biochemistry and Biophysics

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Sterol carrier protein 2 (SCP2) has been investigated by nearly native electrospray ionisation mass spectrometry in the presence of long chain fatty acyl CoAs (LCFA-CoAs) and carnitine derivatives of equivalent fatty acid chain length (LCFA-carnitines). Four SCP2 constructs were compared to examine the influence of the N-terminal presequence and the C-terminal peroxisomal targeting signal on ligand binding. Removal of N-or C-terminal residues did not influence ligand binding. The observation that LCFA-CoAs are high affinity ligands for SCP2 was confirmed, while LCFA-carnitines were demonstrated for the first time not to interact with SCP2. LCFACoAs formed non-covalent complexes with SCP2 of 2:1 and 1:1 stoichiometry, which could be dissociated by elevating the energy of the ions upon entrance to the mass spectrometer. A fluorescence-competition assay using Nile Red butyric acid confirmed the mass spectrometric observations in solution. The physiological significance of the lack of LCFA-carnitine binding by SCP2 is discussed.

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Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network

Cited by 992 publications

References 0 publications

The redox-switch domain of Hsp33 functions as dual stress sensor

The redox-switch domain of Hsp33 functions as dual stress sensor

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

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