Wheel and Deal in the Mitochondrial Inner Membranes: The Tale of Cytochrome c and Cardiolipin

Cardiolipin oxidation and degradation by different factors under severe cell stress serve as a trigger for genetically encoded cell death programs. In this context, the interplay between cardiolipin and another mitochondrial factor—cytochrome c—is a key process in the early stages of apoptosis, and it is a matter of intense research. Cytochrome c interacts with lipid membranes by electrostatic interactions, hydrogen bonds, and hydrophobic effects. Experimental conditions (including pH, lipid composition, and post-translational modifications) determine which specific amino acid residues are involved in the interaction and influence the heme iron coordination state. In fact, up to four binding sites (A, C, N, and L), driven by different interactions, have been reported. Nevertheless, key aspects of the mechanism for cardiolipin oxidation by the hemeprotein are well established. First, cytochrome c acts as a pseudoperoxidase, a process orchestrated by tyrosine residues which are crucial for peroxygenase activity and sensitivity towards oxidation caused by protein self-degradation. Second, flexibility of two weakest folding units of the hemeprotein correlates with its peroxidase activity and the stability of the iron coordination sphere. Third, the diversity of the mode of interaction parallels a broad diversity in the specific reaction pathway. Thus, current knowledge has already enabled the design of novel drugs designed to successfully inhibit cardiolipin oxidation.

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Functional and structural characterization of the myoglobin from the polychaete Ophelia bicornis

Biochemical Journal ◽

10.1042/bj20050267 ◽

2005 ◽

Vol 389 (2) ◽

pp. 497-505 ◽

Cited By ~ 4

Author(s):

M. Teresa Sanna ◽

Barbara Manconi ◽

Massimo Castagnola ◽

Bruno Giardina ◽

Daniela Masia ◽

...

Keyword(s):

Oxygen Affinity ◽

Sperm Whale ◽

Protein Sequencing ◽

Oxygen Binding ◽

Amino Acid Residues ◽

Experimental Conditions ◽

The Stability ◽

Sperm Whale Myoglobin

The myoglobin of the polychaete annelid Ophelia bicornis was isolated, purified to homogeneity and characterized. The primary structure, obtained from cDNA and protein sequencing, consists of 139 amino acid residues. The alignment with other globin sequences showed that O. bicornis myoglobin misses the pre-A helix and the first six residues of the A helix. The presence of a PheB10-GlnE7 haem distal residue pair is in agreement with the measured oxygen affinity (P50=0.85 mmHg; 1 mmHg=0.133 kPa) and the only slightly higher autoxidation rate constant (0.28 h−1) with respect to that of the sperm whale myoglobin mutant E7 His→Gln (0.21 h−1) and to elephant myoglobin (0.1 h−1). Oxygen-binding co-operativity was found to be absent under all the examined experimental conditions. The resistance of O. bicornis myoglobin towards autoxidation seems to confirm the important role of part of the A helix in the stability of the globin. The higher pKa of the acid–alkaline ferric transition of O. bicornis with respect to Asian elephant myoglobin, as well as the higher absorbance ratio of its ferric form to the oxy form measured in the Soret region (γmet/γoxy) with respect to that of the African elephant myoglobin, suggested a stronger interaction between the distal glutamine and the water molecule at the sixth co-ordinate position.

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Revisiting the Self-Assembly of Highly Aromatic Phenylalanine Homopeptides

Molecules ◽

10.3390/molecules25246037 ◽

2020 ◽

Vol 25 (24) ◽

pp. 6037

Author(s):

Enric Mayans ◽

Carlos Alemán

Keyword(s):

Self Assembly ◽

Electrostatic Interactions ◽

Solvent Mixture ◽

Amino Acid Residues ◽

Experimental Conditions ◽

Ordered Nanostructures ◽

Amyloid Aβ ◽

Β Amyloid ◽

First Time ◽

Influence Of Solvent

Diphenylalanine peptide (FF), which self-assembles into rigid tubular nanostructures, is a very short core recognition motif in Alzheimer’s disease β-amyloid (Aβ) polypeptide. Moreover, the ability of the phenylalanine (F or Phe)-homopeptides to self-assemble into ordered nanostructures has been proved. Within this context it was shown that the assembly preferences of this family of compounds is altered by capping both the N- and C-termini using highly aromatic fluorenyl groups (i.e., fluorenyl-9-methoxycarbonyl and 9-fluorenylmethyl ester, named Fmoc and OFm, respectively). In this article the work performed in the field of the effect of the structure and incubation conditions on the morphology and polymorphism of short (from two to four amino acid residues) Phe-homopeptides is reviewed and accompanied by introducing some new results for completing the comparison. Special attention has been paid to the influence of solvent: co-solvent mixture used to solubilize the peptide, the peptide concentration and, in some cases, the temperature. More specifically, uncapped (FF, FFF, and FFFF), N-capped with Fmoc (Fmoc-FF, Fmoc-FFF, and Fmoc-FFFF), C-capped with OFm (FF-OFm), and doubly capped (Fmoc-FF-OFm, Fmoc-FFF-OFm, and Fmoc-FFFF-OFm) Phe-homopeptides have been re-measured. Although many of the experienced assembly conditions have been only revisited as they were previously reported, other experimental conditions have been examined by the first time in this work. In any case, pooling the effect of highly aromatic blocking groups in a single study, using a wide variety of experimental conditions, allows a perspective of how the disappearance of head-to-tail electrostatic interactions and the gradual increase in the amount of π–π stacking interactions, affects the morphology of the assemblies. Future technological applications of Phe-homopeptides can be envisaged by choosing the most appropriate self-assemble structure, defining not only the length of the peptide but also the amount and the position of fluorenyl capping groups.

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Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

10.26434/chemrxiv.9461462 ◽

2019 ◽

Author(s):

Christopher John ◽

Greg M. Swain ◽

Robert P. Hausinger ◽

Denis A. Proshlyakov

Keyword(s):

Active Site ◽

Structural Model ◽

Heme Iron ◽

Chemical Transformations ◽

Experimental Conditions ◽

Strongly Coupled ◽

Non Heme Iron ◽

Reversible Redox ◽

Wide Range ◽

Complex Structural

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an <i>in situ</i> structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semi-empirical computational methods, demonstrating that the Fe (III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and 171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

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Political aspects of security of the European Union Member States

10.31338/1641-2478pe.3.20.6 ◽

2020 ◽

pp. 97-105

Author(s):

Aleksandra Kusztykiewicz-Fedurek

Keyword(s):

European Union ◽

Negative Impact ◽

The European Union ◽

Member States ◽

Eu Member States ◽

Political Security ◽

New Institutions ◽

The Stability ◽

Key Aspects ◽

The Eu

Political security is very often considered through the prism of individual states. In the scholar literature in-depth analyses of this kind of security are rarely encountered in the context of international entities that these countries integrate. The purpose of this article is to draw attention to key aspects of political security in the European Union (EU) Member States. The EU as a supranational organisation, gathering Member States first, ensures the stability of the EU as a whole, and secondly, it ensures that Member States respect common values and principles. Additionally, the EU institutions focus on ensuring the proper functioning of the Eurozone (also called officially “euro area” in EU regulations). Actions that may have a negative impact on the level of the EU’s political security include the boycott of establishing new institutions conducive to the peaceful coexistence and development of states. These threats seem to have a significant impact on the situation in the EU in the face of the proposed (and not accepted by Member States not belonging to the Eurogroup) Eurozone reforms concerning, inter alia, appointment of the Minister of Economy and Finance and the creation of a new institution - the European Monetary Fund.

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Applicability of Instability Index for In vitro Protein Stability Prediction

Protein and Peptide Letters ◽

10.2174/0929866526666190228144219 ◽

2019 ◽

Vol 26 (5) ◽

pp. 339-347 ◽

Cited By ~ 4

Author(s):

Dilani G. Gamage ◽

Ajith Gunaratne ◽

Gopal R. Periyannan ◽

Timothy G. Russell

Keyword(s):

Protein Stability ◽

Caulobacter Crescentus ◽

Effect Of Temperature ◽

Instability Index ◽

Dipeptide Composition ◽

Experimental Conditions ◽

The Stability ◽

Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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Ultrafiltration Method for Plasma Protein Binding Studies and Its Limitations

Processes ◽

10.3390/pr9020382 ◽

2021 ◽

Vol 9 (2) ◽

pp. 382

Author(s):

Camelia-Maria Toma ◽

Silvia Imre ◽

Camil-Eugen Vari ◽

Daniela-Lucia Muntean ◽

Amelia Tero-Vescan

Keyword(s):

Protein Binding ◽

Plasma Protein Binding ◽

Plasma Protein ◽

Specific Binding ◽

Critical Role ◽

Volume Ratio ◽

Binding Studies ◽

Experimental Conditions ◽

Key Aspects

Plasma protein binding plays a critical role in drug therapy, being a key part in the characterization of any compound. Among other methods, this process is largely studied by ultrafiltration based on its advantages. However, the method also has some limitations that could negatively influence the experimental results. The aim of this study was to underline key aspects regarding the limitations of the ultrafiltration method, and the potential ways to overcome them. The main limitations are given by the non-specific binding of the substances, the effect of the volume ratio obtained, and the need of a rigorous control of the experimental conditions, especially pH and temperature. This review presents a variety of methods that can hypothetically reduce the limitations, and concludes that ultrafiltration remains a reliable method for the study of protein binding. However, the methodology of the study should be carefully chosen.

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Effective Separation of Prime Olefins from Gas Stream Using Anion Pillared Metal Organic Frameworks: Ideal Adsorbed Solution Theory Studies, Cyclic Application and Stability

Catalysts ◽

10.3390/catal11040510 ◽

2021 ◽

Vol 11 (4) ◽

pp. 510

Author(s):

Majeda Khraisheh ◽

Fares. Almomani ◽

Gavin Walker

Keyword(s):

Breakthrough Curves ◽

Isotherm Models ◽

Regeneration Ability ◽

Solution Theory ◽

Experimental Conditions ◽

Ideal Adsorbed Solution Theory ◽

Adsorbed Solution ◽

The Stability ◽

Adsorption Desorption ◽

Adsorbed Solution Theory

The separation of C3H4/C3H6 is one of the most energy intensive and challenging operations, requiring up to 100 theoretical stages, in traditional cryogenic distillation. In this investigation, the potential application of two MOFs (SIFSIX-3-Ni and NbOFFIVE-1-Ni) was tested by studying the adsorption–desorption behaviors at a range of operational temperatures (300–360 K) and pressures (1–100 kPa). Dynamic adsorption breakthrough tests were conducted and the stability and regeneration ability of the MOFs were established after eight consecutive cycles. In order to establish the engineering key parameters, the experimental data were fitted to four isotherm models (Langmuir, Freundlich, Sips and Toth) in addition to the estimation of the thermodynamic properties such as the isosteric heats of adsorption. The selectivity of the separation was tested by applying ideal adsorbed solution theory (IAST). The results revealed that SIFSIX-3-Ni is an effective adsorbent for the separation of 10/90 v/v C3H4/C3H6 under the range of experimental conditions used in this study. The maximum adsorption reported for the same combination was 3.2 mmolg−1. Breakthrough curves confirmed the suitability of this material for the separation with a 10-min gab before the lighter C3H4 is eluted from the column. The separated C3H6 was obtained with a 99.98% purity.

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Relative versus absolute RNA quantification: a comparative analysis based on the example of endothelial expression of vasoactive receptors

Biological Procedures Online ◽

10.1186/s12575-021-00144-w ◽

2021 ◽

Vol 23 (1) ◽

Author(s):

Kevin Kuhlmann ◽

Melanie Cieselski ◽

Julia Schumann

Keyword(s):

Dynamic Range ◽

Genetic Material ◽

Housekeeping Genes ◽

Digital Pcr ◽

Absolute Quantification ◽

Test Procedures ◽

Experimental Conditions ◽

Rna Quantification ◽

The Stability ◽

Inflammatory Milieu

Abstract Background In the present study, two distinct PCR methods were used for the quantification of genetic material and their results were compared: real-time-PCR (qPCR; relative quantification) and droplet digital PCR (ddPCR; absolute quantification). The comparison of the qPCR and the ddPCR was based on a stimulation approach of microvascular endothelial cells in which the effect of a pro-inflammatory milieu on the expression of vasoactive receptors was investigated. Results There was consistency in directions of effects for the majority of genes tested. With regard to the indicated dimension of the effects, the overall picture was more differentiated. It was striking that deviations were more pronounced if the measured values were on the extreme edges of the dynamic range of the test procedures. Conclusions To obtain valid and reliable results, dilution series are recommended, which should be carried out initially. In case of ddPCR the number of copies per µl should be adjusted to the low three-digit range. With regard to qPCR it is essential that the stability and reliability of the reference genes used is guaranteed. Here, ddPCR offers the advantage that housekeeping genes are not required. Furthermore, an absolute quantification of the sample can be easily performed by means of ddPCR. Before using ddPCR, however, care should be taken to optimize the experimental conditions. Strict indications for this methodology should also be made with regard to economic and timing factors.

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Effective replacement of cetyltrimethylammonium bromide (CTAB) by mercaptoalkanoic acids on gold nanorod (AuNR) surfaces in aqueous solutions

Nanoscale ◽

10.1039/c9nr09137h ◽

2020 ◽

Vol 12 (2) ◽

pp. 658-668 ◽

Cited By ~ 6

Author(s):

Rafael del Caño ◽

Jose M. Gisbert-González ◽

Jose González-Rodríguez ◽

Guadalupe Sánchez-Obrero ◽

Rafael Madueño ◽

...

Keyword(s):

Aqueous Solutions ◽

Gold Nanorods ◽

Ligand Exchange ◽

Cetyltrimethylammonium Bromide ◽

Gold Nanorod ◽

Experimental Conditions ◽

Seed Mediated ◽

The Stability

The highly packed cetyltrimethylammonium bromide bilayer on the surface of gold nanorods synthesized by the seed-mediated procedure hampers the complete ligand exchange under experimental conditions that preserves the stability of the dispersions.

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Investigations of ternary complexes of Co(II) and Ni(II) with thiodiacetate anion and 1,10-phenanthroline or 2,2’-bipyridine in aqueous solutions

Open Chemistry ◽

10.1515/chem-2015-0048 ◽

2014 ◽

Vol 13 (1) ◽

Author(s):

Dariusz Wyrzykowski ◽

Joanna Pranczk ◽

Dagmara Jacewicz ◽

Aleksandra Tesmar ◽

Bogusław Pilarski ◽

...

Keyword(s):

Aqueous Solutions ◽

Substitution Reactions ◽

Experimental Conditions ◽

Kinetic Measurements ◽

Hydroxo Complexes ◽

Binary Complexes ◽

Vis Spectroscopy ◽

Potentiometric Titration Method ◽

The Stability ◽

Ph Range

AbstractA potentiometric titration method (PT) and a stopped-flow kinetic technique monitored by a UV−Vis spectroscopy have been used to characterize the stability of series of Co(II)- and Ni(II)-thiodiacetato complexes, M(TDA), in the presence of 1,10-phenanthroline (phen) or 2,2’-bipyridine (bipy) in aqueous solutions. The stability constants of the binary (1:1), ternary (1:1:1) as well as the resulting hydroxo complexes were evaluated and compared to the corresponding oxydiacetate complexes. Based on the species distribution as a function of pH the relative predominance of the species in the system over a pH range was discussed. Furthermore, the kinetic measurements of the substitution reactions of the aqua ligands to phen or bipy in the coordination sphere of the binary complexes M(TDA) were performed in the 288–303 K temperature range, at a constant concentration of phen or bipy and at seven different concentrations of the binary complexes (0.2–0.5 mM). The kinetic stability of the M(TDA) complexes was discussed in relation to the experimental conditions and the kind of the auxiliary ligands (phen/bipy). Moreover, the influence of the type of primary ligand (thiodiacetate/oxydiacetate) on the substitution rate of the auxiliary ligands was also compared.

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