Photophysics of the single tryptophan residue in Fusarium solani Cutinase: Evidence for the occurrence of conformational substates with unusual fluorescence behaviour

Weisenborn, Petra C.M.; Meder, Hans; Egmond, Maarten R.; Visser, T.; Hoek, A. van

doi:10.1016/0301-4622(95)00079-8

Cited by 33 publications

(39 citation statements)

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“…Disulphide bridges are known to be excellent quenchers of excited-state aromatic residues. However, selective prolonged irradiation of the Trp residue at 295 nm increases its fluorescence quantum yield, in agreement with previous results by Weisenborn et al (1996) and Prompers et al (1999). It was also observed that on irradiation of cutinase with light at 295 nm, free thiol groups are present in cutinase (see Fig.…”

Section: Discussionsupporting

confidence: 81%

“…Our work reports a much faster process than that already reported by Weisenborn et al (1996). In general, rate of fluorescence signal increase depends on excitation light intensity, protein volume, and diffusion rate (mixing).…”

Section: Theoretical Model For Two-state Chemical Exchangecontrasting

confidence: 49%

“…Assuming five lifetimes did not significantly lower the value of the square root variance. Therefore, four lifetimes were assumed to be consistent with our experimental data, in contrast to Weisenborn et al (1996), who reported three lifetimes.…”

Section: Frequency Domain Intensity Decay Measurementsmentioning

confidence: 51%

“…A partial answer on the effect of UV light on the fungal lipase/esterase cutinase from Fusarium solani pisi has been provided by Weisenborn et al (1996) and Prompers et al (1999). Weisenborn et al (1996) reported that the fluorescence quantum yield of cutinase increases with the illumination time of cutinase, and that the short lifetime component, ∼40 ps, observed for Trp before extended illumination is substituted by longer lifetimes on prolonged UV irradiation of cutinase. The UV illumination has been reported to not affect cutinase lipolytic activity, in contradiction with our findings.…”

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confidence: 99%

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High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

et al. 2002

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It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.Keywords: tryptophan fluorescence lifetime; fluorescence quenching; disulphide (disulfide) bridges; photochemical reaction; protein structure damaged by UV light; SS bond disruption; indole; theoretical model Two divergent theories of the mechanisms involved in ultraviolet (UV) inactivation of enzymes have been developed over a period of years. One theory proposes that the random destruction of any amino acid residue causes inactivation (Augenstein and Riley 1964). The second emphasizes the importance of the disruption of a cluster of specific cystine residues (cysteines involved in disulphide bridges; Augenstein and Riley 1964). It was also found that the effective destruction of cystine, tryptophan, tyrosine, and phenylalanine occurs on UV irradiation of proteins (Kazutomo

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Section: Discussionsupporting

confidence: 81%

Section: Theoretical Model For Two-state Chemical Exchangecontrasting

confidence: 49%

Section: Frequency Domain Intensity Decay Measurementsmentioning

confidence: 51%

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High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

et al. 2002

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“…Apparently, the £uorescence of Trp 69 is quenched by excited state electron transfer to the nearby cystine moiety, which is known to be a very strong quencher [32]. This was already suggested in [1]. The electron capture starts the reaction leading to the reduction of the cystine to a cysteine pair, which still quenches the £uorescence but much less e¤ciently [32].…”

Section: Discussionmentioning

confidence: 89%

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase

Prompers¹,

Hilbers²,

Pepermans³

1999

FEBS Letters

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The fluorescence signal of the single tryptophan residue (Trp 69 ) of Fusarium solani pisi cutinase is highly quenched. However, prolonged irradiation of the enzyme in the tryptophan absorption band causes an increase of the tryptophan fluorescence quantum yield by an order of magnitude. By using a combination of NMR spectroscopy and chemical detection of free thiol groups with a sulfhydryl reagent we could unambiguously show that the unusual fluorescence behaviour of Trp 69 in cutinase is caused by the breaking of the disulfide bond between Cys 31 and Cys 109 upon irradiation, while the amide-aromatic hydrogen bond between Ala 32 and Trp 69 remains intact. This is the first example of tryptophan mediated photoreduction of a disulfide bond in proteins.z 1999 Federation of European Biochemical Societies.

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Deactivation and conformational changes of cutinase in reverse micelles

Melo

Carvalho²,

Aires-Barros³

et al. 1998

Biotechnol. Bioeng.

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Deactivation data and fluorescence intensity changes were used to probe functional and structural stability of cutinase in reverse micelles. A fast deactivation of cutinase in anionic (AOT) reverse micelles occurs due to a reversible denaturation process. The deactivation and denaturation of cutinase is slower in small cationic (CTAB/1‐hexanol) reverse micelles and does not occur when the size of the cationic reverse micellar water‐pool is larger than cutinase. In both systems, activity loss and denaturation are coupled processes showing the same trend with time. Denaturation is probably caused by the interaction between the enzyme and the surfactant interface of the reversed micelle. When the size of the empty reversed micelle water‐pool is smaller than cutinase (at W0 5, with W0 being the water:surfactant concentration ratio) a three‐state model describes denaturation and deactivation with an intermediate conformational state existing on the path from native to denaturated cutinase. This intermediate was clearly detected by an increase in activity and shows only minor conformational changes relative to the native state. At W0 20, the size of the empty water‐pool was larger than cutinase and the data was well described by a two‐state model for both anionic and cationic reverse micelles. For AOT reverse micelles at W0 20, the intermediate state became a transient state and the deactivation and denaturation were described by a two‐state model in which only native and denaturated cutinase were present. For CTAB/1‐hexanol reverse micelles at W0 20, the native cutinase was in equilibrium with an intermediate state, which did not suffer denaturation. 1‐Hexanol showed a stabilizing effect on cutinase in reverse micelles, contributing to the higher stabilities observed in the cationic CTAB/1‐hexanol reverse micelles. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:380–386, 1998.

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Photophysics of the single tryptophan residue in Fusarium solani Cutinase: Evidence for the occurrence of conformational substates with unusual fluorescence behaviour

Cited by 33 publications

References 15 publications

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

Tryptophan mediated photoreduction of disulfide bond causes unusual fluorescence behaviour of Fusarium solani pisi cutinase

Deactivation and conformational changes of cutinase in reverse micelles

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