Effect of Denaturants on the Structural Properties of Soybean Lipoxygenase-1

Malvezzi-Campeggi, Flaminia; Rosato, Nicola; Finazzi‐Agrò, Alessandro; Maccarrone, Mauro

doi:10.1006/bbrc.2001.6109

Cited by 7 publications

(2 citation statements)

References 36 publications

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“…F 1 was greater than F 2 ). This result rules out the possibility that the short-lived component was essentially due to tryptophans located in the N-terminal domain of Lox-1, as previously suggested by urea unfolding measurements (35). The average lifetimes, evaluated from the distribution analysis of Lox-1 and mini-Lox, were Lox-1 Ϸ 1.18 ns and mini-Lox Ϸ 1.00 ns, respectively, indicating a 20% more efficient dynamic quenching in the case of mini-Lox.…”

Section: Resultssupporting

confidence: 85%

“…The fluorescence decay of large, multi-tryptophan-containing proteins is heterogeneous and generally best described by continuous distributions of lifetimes, rather than by few discrete components (42)(43)(44). Previous measurements have shown that the dynamic fluorescence of Lox-1 may be resolved into a pair of lorentzian lifetimes distributions, probably associated to two distinct classes of tryptophylic residues, depending on their relative exposure to the solvent molecules (35). In the present study the phase and demodulation technique (18) demonstrate that in both cases a double distribution is required to fit the experimental data.…”

Section: Resultsmentioning

confidence: 99%

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Structure-to-Function Relationship of Mini-Lipoxygenase, a 60-kDa Fragment of Soybean Lipoxygenase-1 with Lower Stability but Higher Enzymatic Activity

Venere

Salucci

Zadelhoff

et al. 2003

Journal of Biological Chemistry

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Lipoxygenase-1 (Lox-1) is a member of the lipoxygenase family, a class of dioxygenases that take part in the metabolism of polyunsatured fatty acids in eukaryotes. Tryptic digestion of soybean Lox-1 is known to produce a 60 kDa fragment, termed "mini-Lox," which shows enhanced catalytic efficiency and higher membranebinding ability than the native enzyme (Maccarrone, M., Salucci, M. L., van Zadelhoff, G., Malatesta, F., Veldink, G. Vliegenthart, J. F. G., and Finazzi-Agrò , A. (2001) Biochemistry 40, 6819 -6827). In this study, we have investigated the stability of mini-Lox in guanidinium hydrochloride and under high pressure by fluorescence and circular dichroism spectroscopy. Only a partial unfolding could be obtained at high pressure in the range 1-3000 bar at variance with guanidinium hydrochloride. However, in both cases a reversible denaturation was observed. The denaturation experiments demonstrate that mini-Lox is a rather unstable molecule, which undergoes a two-step unfolding transition at moderately low guanidinium hydrochloride concentration (0 -4.5 M). Both chemical-and physical-induced denaturation suggest that mini-Lox is more hydrated than Lox-1, an observation also confirmed by 1-anilino-8-naphthalenesulfonate (ANS) binding studies. We have also investigated the occurrence of substrate-induced changes in the protein tertiary structure by dynamic fluorescence techniques. In particular, eicosatetraynoic acid, an irreversible inhibitor of lipoxygenase, has been used to mimic the effect of substrate binding. We demonstrated that mini-Lox is indeed characterized by much larger conformational changes than those occurring in the native Lox-1 upon binding of eicosatetraynoic acid. Finally, by both activity and fluorescence measurements we have found that 1-anilino-8-naphthalenesulfonate has access to the active site of mini-Lox but not to that of intact Lox-1. These findings strongly support the hypothesis that the larger hydration of mini-Lox renders this molecule more flexible, and therefore less stable.

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

Structure-to-Function Relationship of Mini-Lipoxygenase, a 60-kDa Fragment of Soybean Lipoxygenase-1 with Lower Stability but Higher Enzymatic Activity

Venere

Salucci

Zadelhoff

et al. 2003

Journal of Biological Chemistry

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View full text Add to dashboard Cite

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Electrospray ionization mass spectrometry of soybean lipoxygenases: N‐terminal acetylation, chemical modification, and solution conformation

et al. 2008

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Electrospray ionization mass spectrometry was used to examine both the covalent structure and solution conformation of the soybean lipoxygenases. The post-translational modifications of two lipoxgyenases were identified as N-terminal acetylations by tandem mass spectrometry of peptides generated by trypsin digestion. The N-terminal sequence suggests that the proteins were substrates for the plant homolog of the N-terminal acetyltransferase complex C in yeast. Analysis of samples of native lipoxygenase-3 produced ions corresponding within experimental error to the mass of the N-acetylated polypeptide and one iron atom. The precision of the measurements was within roughly 100 ppm for the 96,856 Da protein. This made it possible to detect the addition of a single oxygen atom to the enzyme in a chemical modification reaction with cumene hydroperoxide. The acid-induced denaturation of lipoxygenase-3, which was accompanied by nearly complete loss of catalytic activity, was observed below pH 3.5 with the appearance of ions in the mass spectrum derived from the apoprotein. There was no evidence for the loss of iron in the absence of unfolding. Solutions of lipoxygenase-3 incubated in 0.1M acetic acid produced ions with a novel charge state distribution suggesting a unique conformation. Circular dichroism measurements showed that the secondary structure features of the native protein were retained in the new conformation. Dynamic light scattering revealed that the new conformation was not a consequence of protein aggregation as the hydrodynamic radius of lipoxygenase-3 was significantly smaller in acetic acid solution than at pH 7.0. Remarkably, the enzyme incubated in acetic acid retained full catalytic activity.

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Tryptophan-Tryptophan Energy Transfer and Classification of Tryptophan Residues in Proteins Using a Therapeutic Monoclonal Antibody as a Model

et al. 2010

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Intrinsic tryptophan (Trp) fluorescence is often used to determine conformational changes of proteins. The fluorescence of multi-Trp proteins is generally assumed to be additive. This assumption usually holds well if Trp residues are situated at long distances from each other in the absence of any excited state reactions involving these residues and therefore when energy transfer does not occur. Here, we experimentally demonstrate energy transfer among Trp residues and support it by a Master Equation kinetic model applied to a therapeutic monoclonal antibody (mAb). The mAbs are one of the most studied and important biologics for the pharmaceutical industry, and they contain many Trp residues in close proximity. Understanding mAb fluorescence is critical for interpreting fluorescence data and protein-structure relationships. We propose that Trp residues could be categorized into three types of emitters in the mAbs. Experimentally, we categorize them according to solvent accessibility based on dependence of their fluorescence lifetime on the external quencher concentration and their emission wavelength. Theoretically, we categorize with molecular dynamics simulations according to their solvent accessibility. This method of combinatorial mapping of fluorescence characteristics can be utilized to illuminate structural aspects as well as make comparisons of drug formulations for these pharmaceutical proteins.

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Effect of Denaturants on the Structural Properties of Soybean Lipoxygenase-1

Cited by 7 publications

References 36 publications

Structure-to-Function Relationship of Mini-Lipoxygenase, a 60-kDa Fragment of Soybean Lipoxygenase-1 with Lower Stability but Higher Enzymatic Activity

Structure-to-Function Relationship of Mini-Lipoxygenase, a 60-kDa Fragment of Soybean Lipoxygenase-1 with Lower Stability but Higher Enzymatic Activity

Electrospray ionization mass spectrometry of soybean lipoxygenases: N‐terminal acetylation, chemical modification, and solution conformation

Tryptophan-Tryptophan Energy Transfer and Classification of Tryptophan Residues in Proteins Using a Therapeutic Monoclonal Antibody as a Model

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