Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, ...stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective
C
m values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.
Recent studies on the chemical alkaline degradation of ferredoxins have contributed to the hypothesis that linear three-iron centres are commonly observed as degradation intermediates of iron-sulfur ...clusters. In this work we assess the validity of this hypothesis. We studied different proteins containing iron-sulfur clusters, iron-sulfur centres and di-iron centres with respect to their chemical degradation kinetics at high pH, in the presence and absence of exogenous sulfide, to investigate the possible formation of linear three-iron centres during protein unfolding. Our spectroscopic and kinetic data show that in these different proteins visible absorption bands at 530 and 620 nm are formed that are identical to those suggested to arise from linear three-iron centres. Iron release and protein unfolding kinetics show that these bands result from the formation of iron sulfides at pH 10, produced by the degradation of the iron centres, and not from rearrangements leading to linear three-iron centres. Thus, at this point any relevant functional role of linear three-iron centres as cluster degradation intermediates in iron-sulfur proteins remains elusive.
Aqueous preparations of the grass Eleusine indica are used for treating malaria and lung infections. Despite its widespread occurrence and therapeutic potential, little is known about its chemical ...composition. This study reports a common chemical pattern for aqueous extracts of E. indica samples from four different localities, separated from each other by approximately 75 to 1340 km, in a wide variety of abiotic and biotic factors. High-performance liquid chromatography with diode array detection (HPLC-DAD), ultra-performance liquid chromatography with diode array detection and mass spectrometry (UPLC-DAD-MS/MS), and nuclear magnetic resonance (NMR) were the analytical techniques applied to characterize substances from E. indica, from each locality. Principal component analysis (PCA) confirmed that E. indica specimens came from four different localities. However, all of the four populations showed a common peaks pattern. This is the first chemical profile report of E. indica. Moreover, p-coumaric acid and isoschaftoside were characterized for the first time in this species.
The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster 3Fe-4S4Fe-4S ferredoxins. Previous studies have shown that these ferredoxins are ...intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase. In this work, a kinetic and spectroscopic investigation on the alkaline chemical denaturation of the protein was performed in an attempt to elucidate the degradation pathway of the iron-sulfur centers in respect to protein unfolding events. For this purpose we investigated cluster dissociation, iron release and protein unfolding by complementary biophysical techniques. We found that shortly after initial protein unfolding, iron release proceeds monophasically at a rate comparable to that of cluster degradation, and that no typical EPR features of linear three-iron sulfur centers are observed. Further, it was observed that EDTA prevents formation of the transient bands and that sulfide significantly enhances its intensity and lifetime, even after protein unfolding. Altogether, our data suggest that iron sulfides, which are formed from the release of iron and sulfide resulting from cluster degradation during protein unfolding in alkaline conditions, are in fact responsible for the observed intermediate spectral species, thus disproving the hypothesis suggesting the presence of a linear three-iron center intermediate. Kinetic studies monitored by visible, fluorescence and UV second-derivative spectroscopies have elicited that upon initial perturbation of the tertiary structure the iron-sulfur centers start decomposing and that the presence of EDTA accelerates the process. Also, the presence of EDTA lowers the observed melting temperature in thermal ramp experiments and the midpoint denaturant concentration in equilibrium chemical unfolding experiments, further suggesting that the clusters also play a structural role in the maintenance of the conformation of the folded state.
Rieske proteins and Rieske ferredoxins are present in the three domains of life and are involved in a variety of cellular processes. Despite their functional diversity, these small Fe–S proteins ...contain a highly conserved all-β fold, which harbors a 2Fe–2S Rieske center. We have identified a novel subtype of Rieske ferredoxins present in hyperthermophilic archaea, in which a two-cysteine conserved SKTPCX
(2–3)
C motif is found at the C-terminus. We establish that in the
Acidianus ambivalens
representative, Rieske ferredoxin 2 (RFd2), these cysteines form a novel disulfide bond within the Rieske fold, which can be selectively broken under mild reducing conditions insufficient to reduce the 2Fe–2S cluster or affect the secondary structure of the protein, as shown by visible circular dichroism, absorption, and attenuated total reflection Fourier transform IR spectroscopies. RFd2 presents all the EPR, visible absorption, and visible circular dichroism spectroscopic features of the 2Fe–2S Rieske center. The cluster has a redox potential of +48 mV (25 °C and pH 7) and a p
K
a
of 10.1 ± 0.2. These shift to +77 mV and 8.9 ± 0.3, respectively, upon reduction of the disulfide. RFd2 has a melting temperature near the boiling point of water (
T
m
= 99 °C, pH 7.0), but it becomes destabilized upon disulfide reduction (Δ
T
m
= −9 °C, Δ
C
m
= −0.7 M guanidinium hydrochloride). This example illustrates how the incorporation of an additional structural element such as a disulfide bond in a highly conserved fold such as that of the Rieske domain may fine-tune the protein for a particular function or for increased stability.
Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca2+ ...dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca2+. We show that at physiological pH, Ca2+ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca2+ boosts the onset of SOD1 aggregation. In agreement, Ca2+ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca2+ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca2+-induced aggregates, thus indicating that Ca2+ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca2+ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca2+ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.
Background: SOD1-enriched protein inclusions and Ca2+ overload are hallmarks in ALS-affected motor neurons. Ca2+ burden correlates with SOD1 aggregation in cellular models.
Results: Ca2+ induces conformational changes that enhance and shift SOD1 aggregation from fibrils toward amorphous aggregates.
Conclusion: SOD1 aggregation is enhanced and modulated by Ca2+.
Significance: Ca2+ can behave as a pathogenic effector in the formation of ALS proteinaceous inclusions.
Detailed structural models of di-cluster seven-iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors’ role in protein stability. The here ...reported, hemihedric twinned crystal structure at 2.0
Å resolution from
Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N-terminal extension comprising a His/Asp Zn
2+ site and the ferredoxin (βαβ)
2 core, which harbours intact clusters I and II, a 3Fe–4S
1+/0 and a 4Fe–4S
2+/1+ centres. This is in contrast with the previously available ferredoxin structure from
Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two 3Fe–4S
1+/0 centres and poor definition around cluster II.
Detailed structural models of di‐cluster seven‐iron ferredoxins constitute a valuable resource for folding and stability studies relating the metal cofactors’ role in protein stability. The here ...reported, hemihedric twinned crystal structure at 2.0 Å resolution from Acidianus ambivalens ferredoxin, shows an integral 103 residues, physiologically relevant native form composed by a N‐terminal extension comprising a His/Asp Zn2+ site and the ferredoxin (βαβ)2 core, which harbours intact clusters I and II, a 3Fe–4S1+/0 and a 4Fe–4S2+/1+ centres. This is in contrast with the previously available ferredoxin structure from Sulfolofus tokodai, which was obtained from an artificial oxidative conversion with two 3Fe–4S1+/0 centres and poor definition around cluster II.
Corynebacterium diphtheriae strains displayed different degrees of attachment to HEp-2 cell monolayers with two distinct adherence patterns, termed localised (LA) and diffuse (DA). The LA phenotype ...predominated over the DA phenotype. The non-sucrose fermenting strains expressing DA pattern adhered mostly with high index values (≥10
bact/cell). Low adhesion index (<10
bact/cell) was mainly observed among sucrose fermenting strains. The fluorescein isothiocyanate (FITC)-labelled phalloidin assay (fluorescent-actin staining test) showed positive results for microorganisms of both LA and DA phenotypes. The FITC-labelled
C. diphtheriae non-fimbrial surface proteins 67–72p interacted directly with HEp-2 cell membranes. Therefore, toxigenic
C. diphtheriae exhibited LA and DA adherence patterns and ability to induce actin polymerisation. The experimental evidences also pointed to 67–72p as putative adhesins of
C. diphtheriae to HEp-2 cells.
•Cortisol detection based on surface plasmon resonance (SPR) using an unclad plastic optical fiber (POF).•High sensitivity and low limit of detection (LOD).•Selectivity of the proposed sensor for ...stress hormone was performed showing high specificity.•Optical sensor solution with relatively low-cost interrogation method, straightforward signal processing.•Interesting working range (0.005−10 ng/mL) for different biological samples from human or marine life.
This paper presents the development and feasibility tests of a cortisol immunosensor. The sensor is based on surface plasmon resonance (SPR) using an unclad plastic optical fiber (POF) in which the SPR is used as sensitivity enhancer, promoted by a gold/palladium (AuPd) alloy coating. The AuPd coated fibers were functionalized with an anti-cortisol antibody and passivated with bovine serum albumin (BSA) to be tested in the presence of cortisol as target analyte. The antibody-antigen binding reaction caused a variation of the refractive index on the surface of the AuPd coating, which leads to a shift of the SPR signature wavelength. The sensor was tested for different cortisol concentrations, ranging from 0.005 to 10 ng/mL. The reported biosensor presented a total wavelength shift of 15 nm for the testing range, putting in evidence a high sensitivity. Control tests for selectivity assessment were also performed. Concentrations as high as 10 ng/mL of cortisol, in a sensor functionalized with anti-hCG antibodies, only resulted in 1 nm variation of the resonance wavelength, 15 times lower than the one functionalized with the anti-cortisol antibodies, which indicates a high selectivity for the proposed approach. For this sensing approach the limit of detection (LOD) was determined to be 1 pg/mL. The proposed SPR based POF sensor has a low-cost interrogation method, high sensitivity and low LOD, straightforward signal processing and find important applications in different biological fields.
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