Conformational States in Denaturants of Cytochrome <i>c</i> and Horseradish Peroxidases Examined by Fluorescence and Circular Dichroism

Tsaprailis, George; Chan, Doreen Wing Sze; English, Ann M.

doi:10.1021/bi971032a

Cited by 69 publications

(81 citation statements)

References 71 publications

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“…Using circular dichroism (CD) and a commercial HRP preparation similar to ours, Tsaprailis et al (1998) noted a single-step loss of HRP secondary structure between 1.2 and 2.7 M GdCl, with complete dissociation of the heme at 2.6 M GdCl and fluorescence changes in Trp117 above 4 M GdCl at pH 7.0. The unfolding half-time of native HRP in 6 M GdCl was 8.65 min.…”

Section: Unfolding Of Native and Pa-hrpsupporting

confidence: 51%

“…This difference could possibly arise from the in- troduction of a negative charge by phthalic anhydride modification close to one of the Ca 2+ binding sites. Tsaprailis et al (1998) observed that prior incubation with 2 mM EDTA for 18 h made HRP more prone to GdCl-induced denaturation and that HRP was greatly destabilized by EDTA combined with a reducing agent (DTT). Here, the reducing agent TCEP cleaves HRP's disulfide bridges in presence of GdCl, leading to unfolding and decreased activity.…”

Section: Unfolding Of Native and Pa-hrpmentioning

confidence: 99%

“…Additional biophysical studies using NMR (Veitch et al, 1995(Veitch et al, , 1997, resonance Raman spectroscopy (Smulevitch et al, 1994), and the availability of recombinant mutants (e.g., Gilfoyle et al, 1996;Rodriguez-Lopez et al, 1996a,b) have already yielded new insights into its structure-function relationships; see Veitch and Smith (2001) for a recent review. Many protein folding and stability studies (e.g., Tsaprailis et al, 1998) have used HRP, which has many biotechnological applications. It acts as an indicator enzyme in "cold" DNA probes (Renz and Kurz, 1984) and in immunoassays (Tijssen, 1985).…”

Section: Introductionmentioning

confidence: 99%

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Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

O'Brien

Smith

Ó’Fágáin

2002

Biotech & Bioengineering

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Phthalic anhydride (PA) modification stabilizes horseradish peroxidase (HRP) by reversal of the positive charge on two of HRP's six lysine residues. Native and PA-HRP had half-inactivation temperatures of 51 and 65 degrees C and half-lives at 65 degrees C of 4 and 17 min, respectively. PA-HRP was more resistant to dimethylformamide at room temperature and tetrahydrofuran at 60 degrees C and to unfolding by heat, guanidine chloride, EDTA, and the reducing agent tris(2-carboxyethyl)phosphine hydrochloride. Binding of the hydrophobic probe Nile Red to the native enzyme and to PA-HRP was similar. The kinetics of both HRPs with the substrates ABTS, ferrocyanide, ferulic acid, and indole-3-propionic acid were measured, as was binding of the inhibitor benzhydroxamic acid. Small improvements in the catalytic properties were detected.

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Section: Unfolding Of Native and Pa-hrpsupporting

confidence: 51%

Section: Unfolding Of Native and Pa-hrpmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

O'Brien

Smith

Ó’Fágáin

2002

Biotech & Bioengineering

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“…Previous in vitro studies showed that active HRP is formed via at least two steps: folding of nascent polypeptide to apoenzyme, which is assisted by calcium, and its conversion to holoenzyme by binding hemin (29,31). We therefore examined the effect of CaCl 2 on folding of HRP in the present study.…”

Section: Overexpression Of Dsb Proteins Alleviates Growth Inhibitionmentioning

confidence: 98%

Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

Kurokawa

Yanagi

Yura

2000

Appl Environ Microbiol

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Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli. By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli. When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli.

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“…The insertion of an additional disulfide bond close to the distal Ca 2ϩ ion-binding site resulted in an apparent increase in the stability of the heme environment and active state of the protein. The particular significance of the Ca 2ϩ ions and disulfide bridges to protein stability has been underlined by a comparative study of HRPC and the class I peroxidase cytochrome c peroxidase in the presence of denaturants (44). The considerably lower kinetic stability found for cytochrome c peroxidase was attributed to the absence of these two stabilizing structural elements.…”

Section: Roles Of the Calcium Ions In Hrpcmentioning

confidence: 99%

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

Howes

Feis

Raimondi³

et al. 2001

Journal of Biological Chemistry

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The extent to which the structural Ca 2؉ ions of horseradish peroxidase (HRPC) are a determinant in defining the heme pocket architecture is investigated by electronic absorption and resonance Raman spectroscopy upon removal of one Ca 2؉ ion. The Fe(III) heme states are modified upon Ca 2؉ depletion, with an uncommon quantum mechanically mixed spin state becoming the dominant species. Ca 2؉ -depleted HRPC forms complexes with benzohydroxamic acid and CO which display spectra very similar to those of native HRPC, indicating that any changes to the distal cavity structural properties upon Ca 2؉ depletion are easily reversed. Contrary to the native protein, the Ca 2؉ -depleted ferrous form displays a low-spin bis-histidyl heme state and a small proportion of high-spin heme. Furthermore, the (Fe-Im) stretching mode downshifts 27 cm ؊1 upon Ca 2؉ depletion revealing a significant structural perturbation of the proximal cavity near the histidine ligand. The specific activity of the Ca 2؉ -depleted enzyme is 50% that of the native form. The effects on enzyme activity and spectral features observed upon Ca 2؉ depletion are reversible upon reconstitution. Evaluation of the present and previous data firmly favors the proximal Ca 2؉ ion as that which is lost upon Ca 2؉ depletion and which likely plays the more critical role in regulating the heme pocket structural and catalytic properties.Peroxidases of the plant peroxidase superfamily are hemecontaining enzymes that oxidize a variety of aromatic molecules in the presence of hydrogen peroxide. They include peroxidases of plant, fungal, and prokaryotic origins that can be divided into three classes based on sequence alignment (1).Additional support for such a classification was gained as the crystal structures of representative enzymes of each class became available. Class I contains bacterial peroxidases and peroxidases from plant mitochondria and chloroplasts, for example most ascorbate peroxidases and cytochrome c peroxidase. Class II contains extracellular fungal peroxidases such as Coprinus cinereus peroxidase (a peroxidase essentially identical to Arthromyces ramosus peroxidase) and lignin-degrading peroxidases. Class III contains secretory plant peroxidases, typified by the classical horseradish peroxidase isoenzyme C (HRPC). The peroxidases of classes II and III share a number of structural elements considered to be of importance for maintaining protein stability and activity. These include calciumbinding sites proximal and distal to the heme and four disulfide bridges (1-5); the latter was in different locations in class II with respect to class III peroxidases. These features are absent in class I peroxidases. Class III peroxidases also have a loop insertion in the sequence, composed of the DЈ, FЈ, and FЉ helices, not found in the other classes. The relationship between calcium binding and enzyme inactivation has been treated primarily by studies on lignin peroxidase (LIP) (6, 7), manganese peroxidase (MNP) (8, 9), and HRPC (10, 11). Thermal (6) and alkaline (7) i...

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Conformational States in Denaturants of Cytochrome c and Horseradish Peroxidases Examined by Fluorescence and Circular Dichroism

Cited by 69 publications

References 71 publications

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

Effects of phthalic anhydride modification on horseradish peroxidase stability and activity

Overexpression of Protein Disulfide Isomerase DsbC Stabilizes Multiple-Disulfide-Bonded Recombinant Protein Produced and Transported to the Periplasm in Escherichia coli

The Critical Role of the Proximal Calcium Ion in the Structural Properties of Horseradish Peroxidase

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