Publications by authors named "Rosario Oliva"

45 Publications

Deep sea osmolytes in action: their effect on protein-ligand binding under high pressure stress.

Phys Chem Chem Phys 2022 Aug 3;24(30):17966-17978. Epub 2022 Aug 3.

Physical Chemistry I - Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Because organisms living in the deep sea and in the sub-seafloor must be able to cope with hydrostatic pressures up to 1000 bar and more, their biomolecular processes, including ligand-binding reactions, must be adjusted to keep the associated volume changes low in order to function efficiently. Almost all organisms use organic cosolvents (osmolytes) to protect their cells from adverse environmental conditions. They counteract osmotic imbalance, stabilize the structure of proteins and maintain their function. We studied the binding properties of the prototypical ligand proflavine to two serum proteins with different binding pockets, BSA and HSA, in the presence of two prominent osmolytes, trimethylamine--oxide (TMAO) and glycine betaine (GB). TMAO and GB play an important role in the regulation and adaptation of life in deep-sea organisms. To this end, pressure dependent fluorescence spectroscopy was applied, supplemented by circular dichroism (CD) spectroscopy and computer modeling studies. The pressure-dependent measurements were also performed to investigate the intimate nature of the complex formation in relation to hydration and packing changes caused by the presence of the osmolytes. We show that TMAO and GB are able to modulate the ligand binding process in specific ways. Depending on the chemical make-up of the protein's binding pocket and thus the thermodynamic forces driving the binding process, there are osmolytes with specific interaction sites and binding strengths with water that are able to mediate efficient ligand binding even under external stress conditions. In the binding of proflavine to BSA and HSA, the addition of both compatible osmolytes leads to an increase in the binding constant upon pressurization, with TMAO being the most efficient, rendering the binding process also insensitive to pressurization even up to 2 kbar as the volume change remains close to zero. This effect can be corroborated by the effects the cosolvents impose on the strength and dynamics of hydration water as well as on the conformational dynamics of the protein.
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http://dx.doi.org/10.1039/d2cp01769eDOI Listing
August 2022

Suppression of Liquid-Liquid Phase Separation and Aggregation of Antibodies by Modest Pressure Application.

Chemistry 2022 Jun 27:e202201658. Epub 2022 Jun 27.

Physical Chemistry I-Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Strasse 4a, 44227, Dortmund, Germany.

The high colloidal stability of antibody (immunoglobulin) solutions is important for pharmaceutical applications. Inert cosolutes, excipients, are generally used in therapeutic protein formulations to minimize physical instabilities, such as liquid-liquid phase separation (LLPS), aggregation and precipitation, which are often encountered during manufacturing and storage. Despite their widespread use, a detailed understanding of how excipients modulate the specific protein-protein interactions responsible for these instabilities is still lacking. In this work, we demonstrate the high sensitivity to pressure of globulin condensates as a suitable means to suppress LLPS and subsequent aggregation of concentrated antibody solutions. The addition of excipients has only a minor effect. The high pressure sensitivity observed is due to the fact that these flexible Y-shaped molecules create a considerable amount of void volume in the condensed phase, leading to an overall decrease in the volume of the system upon dissociation of the droplet phase by pressure already at a few tens of to hundred bar. Moreover, we show that immunoglobulin molecules themselves are highly resistant to unfolding under pressure, and can even sustain pressures up to about 6 kbar without conformational changes. This implies that immunoglobulins are resistant to the pressure treatment of foods, such as milk, in high-pressure food-processing technologies, thereby preserving their immunological activity.
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http://dx.doi.org/10.1002/chem.202201658DOI Listing
June 2022

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex.

Int J Mol Sci 2022 May 19;23(10). Epub 2022 May 19.

Physical Chemistry I-Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.
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http://dx.doi.org/10.3390/ijms23105690DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9145931PMC
May 2022

The C-terminus of the GKY20 antimicrobial peptide, derived from human thrombin, plays a key role in its membrane perturbation capability.

Phys Chem Chem Phys 2022 Mar 30;24(13):7994-8002. Epub 2022 Mar 30.

Department of Chemical Sciences, University of Naples Federico II, Via Cintia 4, 80126 Naples, Italy.

Previously, we characterized in detail the mechanism of action of the antimicrobial peptide GKY20, showing that it selectively perturbs the bacterial-like membrane employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. Here, we used a combination of biophysical techniques to similarly characterize the antimicrobial activity as well as the membrane perturbing capability of GKY10, a much shorter version of the GKY20 peptide. GKY10 is only half of the parent peptide and consists of the last 10 amino acids (starting from the C-terminus) of the full-length peptide. Despite a large difference in length, we found that GKY10, like the parent peptide, retains the ability to adopt a helical structure and to induce lipid segregation upon membrane binding. Overall, our results suggest that the amino acid sequence of GKY10 is responsible for most of the observed behaviors of GKY20. Our results shed further light on the mechanism of action of the full-length peptide and provide useful information for the design and development of new peptides that serve as antimicrobial agents.
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http://dx.doi.org/10.1039/d1cp05857fDOI Listing
March 2022

Effects of Cosolvents and Crowding Agents on the Stability and Phase Transition Kinetics of the SynGAP/PSD-95 Condensate Model of Postsynaptic Densities.

J Phys Chem B 2022 03 16;126(8):1734-1741. Epub 2022 Feb 16.

Physical Chemistry I - Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany.

The SynGAP/PSD-95 binary protein system serves as a simple mimicry of postsynaptic densities (PSDs), which are protein assemblies based largely on liquid-liquid phase separation (LLPS), that are located underneath the plasma membrane of excitatory synapses. Surprisingly, the LLPS of the SynGAP/PSD-95 system is much more pressure sensitive than typical folded states of proteins or nucleic acids. It was found that phase-separated SynGAP/PSD-95 droplets dissolve into a homogeneous solution at a pressure of tens to hundred bar. Since organisms in the deep sea are exposed to pressures of up to ∼1000 bar, this observation suggests that deep-sea organisms must counteract the high pressure sensitivity of PSDs to avoid neurological impairment. We demonstrate here that the compatible osmolyte trimethylamine--oxide (TMAO) as well as macromolecular crowding agents at moderate concentrations can mitigate the deleterious effect of pressure on SynGAP/PSD-95 droplet stability, extending stable LLPS up to almost a kbar level. Moreover, the formation of SynGAP/PSD-95 droplets is a very rapid process that can be switched on and off in seconds. In contrast with the marked effects of the cosolutes on droplet stability, at the cosolutes' respective biologically relevant concentrations, their impact on the phase transformation kinetics is rather small. Only a high TMAO concentration results in a significant kinetic retardation of LLPS. Taken together, these findings offer new biophysical insights into the neurological effects of hydrostatic pressure. In particular, our results indicate how pressure-induced neurological disorders might be alleviated by upregulating certain cosolutes in the cellular milieu.
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http://dx.doi.org/10.1021/acs.jpcb.2c00794DOI Listing
March 2022

Sex Hormone Receptor Expression in Craniopharyngiomas and Association with Tumor Aggressiveness Characteristics.

J Clin Med 2022 Jan 5;11(1). Epub 2022 Jan 5.

Unidad de Gestión de Endocrinología y Nutrición, Instituto de Biomedicina de Sevilla (IBiS), Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, 41013 Sevilla, Spain.

Craniopharyngiomas (CPs) are rare tumors of the sellar and suprasellar regions of embryonic origin. The primary treatment for CPs is surgery but it is often unsuccessful. Although CPs are considered benign tumors, they display a relatively high recurrence rate that might compromise quality of life. Previous studies have reported that CPs express sex hormone receptors, including estrogen and progesterone receptors. Here, we systematically analyzed estrogen receptor α (ERα) and progesterone receptor (PR) expression by immunohistochemistry in a well-characterized series of patients with CP ( = 41) and analyzed their potential association with tumor aggressiveness features. A substantial proportion of CPs displayed a marked expression of PR. However, most CPs expressed low levels of ERα. No major association between PR and ERα expression and clinical aggressiveness features was observed in CPs. Additionally, in our series, β-catenin accumulation was not related to tumor recurrence.
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http://dx.doi.org/10.3390/jcm11010281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8746083PMC
January 2022

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Int J Mol Sci 2021 Dec 10;22(24). Epub 2021 Dec 10.

Department of Biology, University of Naples Federico II, 80126 Naples, Italy.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoB, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and cells. K values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.
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http://dx.doi.org/10.3390/ijms222413312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8706149PMC
December 2021

Ions in the Deep Subsurface of Earth, Mars, and Icy Moons: Their Effects in Combination with Temperature and Pressure on tRNA-Ligand Binding.

Int J Mol Sci 2021 Oct 8;22(19). Epub 2021 Oct 8.

Physical Chemistry I-Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, 44227 Dortmund, Germany.

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions' role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl, MgSO, and Mg(ClO)) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid-ligand interactions of the type studied here.
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http://dx.doi.org/10.3390/ijms221910861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8509373PMC
October 2021

Untangling the interaction of α-synuclein with DNA i-motifs and hairpins by volume-sensitive single-molecule FRET spectroscopy.

RSC Chem Biol 2021 Aug 2;2(4):1196-1200. Epub 2021 Jul 2.

Physical Chemistry I-Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Strasse 4a Dortmund D-44227 Germany

The intrinsically disordered protein α-synuclein causes Parkinson's disease by forming toxic oligomeric aggregates inside neurons. Single-molecule FRET experiments revealed conformational changes of noncanonical DNA structures, such as i-motifs and hairpins, in the presence of α-synuclein. Volumetric analyses revealed differences in binding mode, which is also affected by cellular osmolytes.
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http://dx.doi.org/10.1039/d1cb00108fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341996PMC
August 2021

The Effects of Temperature and Pressure on Protein-Ligand Binding in the Presence of Mars-Relevant Salts.

Biology (Basel) 2021 Jul 20;10(7). Epub 2021 Jul 20.

Physical Chemistry I-Biophysical Chemistry, Department of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Protein-ligand interactions are fundamental to all biochemical processes. Generally, these processes are studied at ambient temperature and pressure conditions. We investigated the binding of the small ligand 8-anilinonaphthalene-1-sulfonic acid (ANS) to the multifunctional protein bovine serum albumin (BSA) at ambient and low temperatures and at high pressure conditions, in the presence of ions associated with the surface and subsurface of Mars, including the chaotropic perchlorate ion. We found that salts such as magnesium chloride and sulfate only slightly affect the protein-ligand complex formation. In contrast, magnesium perchlorate strongly affects the interaction between ANS and BSA at the single site level, leading to a change in stoichiometry and strength of ligand binding. Interestingly, both a decrease in temperature and an increase in pressure favor the ligand binding process, resulting in a negative change in protein-ligand binding volume. This suggests that biochemical reactions that are fundamental for the regulation of biological processes are theoretically possible outside standard temperature and pressure conditions, such as in the harsh conditions of the Martian subsurface.
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http://dx.doi.org/10.3390/biology10070687DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8301423PMC
July 2021

Impact of a Single Point Mutation on the Antimicrobial and Fibrillogenic Properties of Cryptides from Human Apolipoprotein B.

Pharmaceuticals (Basel) 2021 Jun 29;14(7). Epub 2021 Jun 29.

Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy.

Host defense peptides (HDPs) are gaining increasing interest, since they are endowed with multiple activities, are often effective on multidrug resistant bacteria and do not generally lead to the development of resistance phenotypes. Cryptic HDPs have been recently identified in human apolipoprotein B and found to be endowed with a broad-spectrum antimicrobial activity, with anti-biofilm, wound healing and immunomodulatory properties, and with the ability to synergistically act in combination with conventional antibiotics, while being not toxic for eukaryotic cells. Here, a multidisciplinary approach was used, including time killing curves, differential scanning calorimetry, circular dichroism, ThT binding assays, and transmission electron microscopy analyses. The effects of a single point mutation (Pro → Ala in position 7) on the biological properties of ApoB-derived peptide r(P)ApoB have been evaluated. Although the two versions of the peptide share similar antimicrobial and anti-biofilm properties, only r(P)ApoB peptide was found to exert bactericidal effects. Interestingly, antimicrobial activity of both peptide versions appears to be dependent from their interaction with specific components of bacterial surfaces, such as LPS or LTA, which induce peptides to form β-sheet-rich amyloid-like structures. Altogether, obtained data indicate a correlation between ApoB-derived peptides self-assembling state and their antibacterial activity.
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http://dx.doi.org/10.3390/ph14070631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8308739PMC
June 2021

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Chemistry 2021 Aug 22;27(46):11845-11851. Epub 2021 Jul 22.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227, Dortmund, Germany.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.
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http://dx.doi.org/10.1002/chem.202101592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8457056PMC
August 2021

Structural responses of model biomembranes to Mars-relevant salts.

Phys Chem Chem Phys 2021 Jul;23(26):14212-14223

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Lipid membranes are a key component of contemporary living systems and are thought to have been essential to the origin of life. Most research on membranes has focused on situations restricted to ambient physiological or benchtop conditions. However, the influence of more extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments are less well understood. The deep subsurface environments of Mars, for instance, may harbor high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments for cellular life. Here, we investigated the combined effects of high concentrations of salts, including sodium and magnesium perchlorate and sulfate, and high hydrostatic pressure on the stability and structure of model biomembranes of varying complexity. To this end, a variety of biophysical techniques have been applied, which include calorimetry, fluorescence spectroscopies, small-angle X-ray scattering, dynamic light scattering, and microscopy techniques. We show that the structure and phase behavior of lipid membranes is sensitively dictated by the nature of the salt, in particular its anion and its concentration. We demonstrate that, with the exception of magnesium perchlorate, which can also induce cubic lipid arrangements, long-chain saturated lipid bilayer structures can still persist at high salt concentrations across a range of pressures. The lateral organization of complex heterogeneous raft-like membranes is affected by all salts. For simple, in particular bacterial membrane-type bilayer systems with unsaturated chains, vesicular structures are still stable at Martian brine conditions, also up to the kbar pressure range, demonstrating the potential compatibility of environments containing such ionic and pressure extremes to lipid-encapsulated life.
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http://dx.doi.org/10.1039/d1cp02092gDOI Listing
July 2021

The N-terminal domain of the prion protein is required and sufficient for liquid-liquid phase separation: A crucial role of the Aβ-binding domain.

J Biol Chem 2021 07 6;297(1):100860. Epub 2021 Jun 6.

Department of Biochemistry of Neurodegenerative Diseases, Institute of Biochemistry and Pathobiochemistry, Ruhr University Bochum, Bochum, Germany; Cluster of Excellence RESOLV, Ruhr University Bochum, Bochum, Germany. Electronic address:

Formation of biomolecular condensates through liquid-liquid phase separation (LLPS) has been described for several pathogenic proteins linked to neurodegenerative diseases and is discussed as an early step in the formation of protein aggregates with neurotoxic properties. In prion diseases, neurodegeneration and formation of infectious prions is caused by aberrant folding of the cellular prion protein (PrP). PrP is characterized by a large intrinsically disordered N-terminal domain and a structured C-terminal globular domain. A significant fraction of mature PrP is proteolytically processed in vivo into an entirely unstructured fragment, designated N1, and the corresponding C-terminal fragment C1 harboring the globular domain. Notably, N1 contains a polybasic motif that serves as a binding site for neurotoxic Aβ oligomers. PrP can undergo LLPS; however, nothing is known how phase separation of PrP is triggered on a molecular scale. Here, we show that the intrinsically disordered N1 domain is necessary and sufficient for LLPS of PrP. Similar to full-length PrP, the N1 fragment formed highly dynamic liquid-like droplets. Remarkably, a slightly shorter unstructured fragment, designated N2, which lacks the Aβ-binding domain and is generated under stress conditions, failed to form liquid-like droplets and instead formed amorphous assemblies of irregular structures. Through a mutational analysis, we identified three positively charged lysines in the postoctarepeat region as essential drivers of condensate formation, presumably largely via cation-π interactions. These findings provide insights into the molecular basis of LLPS of the mammalian prion protein and reveal a crucial role of the Aβ-binding domain in this process.
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http://dx.doi.org/10.1016/j.jbc.2021.100860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254114PMC
July 2021

Toxicity and membrane perturbation properties of the ribotoxin-like protein Ageritin.

J Biochem 2021 Dec;170(4):473-482

Department of Environmental, Biological and Pharmaceutical Sciences and Technologies (DiSTABiF), University of Campania 'Luigi Vanvitelli', Via Vivaldi 43, 81100 Caserta, Italy.

Ageritin is the prototype of a new ribotoxin-like protein family, which has been recently identified also in basidiomycetes. The protein exhibits specific RNase activity through the cleavage of a single phosphodiester bond located at sarcin/ricin loop of the large rRNA, thus inhibiting protein biosynthesis at early stages. Conversely to other ribotoxins, its activity requires the presence of divalent cations. In the present study, we report the activity of Ageritin on both prokaryotic and eukaryotic cells showing that the protein has a prominent effect on cancer cells viability and no effects on eukaryotic and bacterial cells. In order to rationalize these findings, the ability of the protein to interact with various liposomes mimicking normal, cancer and bacterial cell membranes was explored. The collected results indicate that Ageritin can interact with DPPC/DPPS/Chol vesicles, used as a model of cancer cell membranes, and with DPPC/DPPG vesicles, used as a model of bacterial cell membranes, suggesting a selective interaction with anionic lipids. However, a different perturbation of the two model membranes, mediated by cholesterol redistribution, was observed and this might be at the basis of Ageritin selective toxicity towards cancer cells.
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http://dx.doi.org/10.1093/jb/mvab062DOI Listing
December 2021

Towards DNA-Encoded Micellar Chemistry: DNA-Micelle Association and Environment Sensitivity of Catalysis.

Chemistry 2021 Jul 7;27(39):10048-10057. Epub 2021 Jun 7.

Medicinal Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 6, 44227, Dortmund, Germany.

The development of DNA-compatible reaction methodologies is a central theme to advance DNA-encoded screening library technology. Recently, we were able to show that sulfonic acid-functionalized block copolymer micelles facilitated Brønsted acid-promoted reactions such as the Povarov reaction on DNA-coupled starting materials with minimal DNA degradation. Here, the impact of polymer composition on micelle shape, and reaction conversion was investigated. A dozen sulfonic acid-functionalized block copolymers of different molar mass and composition were prepared by RAFT polymerization and were tested in the Povarov reaction, removal of the Boc protective group, and the Biginelli reaction. The results showed trends in the polymer structure-micellar catalytic activity relationship. For instance, micelles composed of block copolymers with shorter acrylate ester chains formed smaller particles and tended to provide faster reaction kinetics. Moreover, fluorescence quenching experiments as well as circular dichroism spectroscopy showed that DNA-oligomer-conjugates, although highly water-soluble, accumulated very effectively in the micellar compartments, which is a prerequisite for carrying out a DNA-encoded reaction in the presence of polymer micelles.
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http://dx.doi.org/10.1002/chem.202100980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8361662PMC
July 2021

Unraveling the binding characteristics of small ligands to telomeric DNA by pressure modulation.

Sci Rep 2021 05 6;11(1):9714. Epub 2021 May 6.

Physical Chemistry I-Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Strasse 4a, 44227, Dortmund, Germany.

Recently, non-canonical DNA structures, such as G-quadruplexes (GQs), were found to be highly pressure sensitive, suggesting that pressure modulation studies can provide additional mechanistic details of such biomolecular systems. Using FRET and CD spectroscopy as well as binding equilibrium measurements, we investigated the effect of pressure on the binding reaction of the ligand ThT to the quadruplex 22AG in solutions containing different ionic species and a crowding agent mimicking the intracellular milieu. Pressure modulation helped us to identify the different conformational substates adopted by the quadruplex at the different solution conditions and to determine the volumetric changes during complex formation and the conformational transitions involved. The magnitudes of the binding volumes are a hallmark of packing defects and hydrational changes upon ligand binding. The conformational substates of the GQ as well as the binding strength and the stoichiometry of complex formation depend strongly on the solution conditions as well as on pressure. High hydrostatic pressure can also impact GQs inside living cells and thus affect expression of genetic information in deep sea organisms. We show that sub-kbar pressures do not only affect the conformational dynamics and structures of GQs, but also their ligand binding reactions.
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http://dx.doi.org/10.1038/s41598-021-89215-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102477PMC
May 2021

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III.

Int J Mol Sci 2021 Mar 11;22(6). Epub 2021 Mar 11.

Department of Chemical Sciences, University of Naples Federico II, Via Cintia, 4, 80126 Naples, Italy.

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee . LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl--glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl--glycero-3--phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.
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http://dx.doi.org/10.3390/ijms22062857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001998PMC
March 2021

Covalently bonded hopanoid-Lipid A from Bradyrhizobium: The role of unusual molecular structure and calcium ions in regulating the lipid bilayers organization.

J Colloid Interface Sci 2021 Jul 18;594:891-901. Epub 2021 Mar 18.

CSGI, Center for Colloid and Surface Science, via della Lastruccia 3, Sesto Fiorentino (FI), Italy; Department of Chemical Sciences, University of Naples Federico II, via Cintia 4, 80126 Naples, Italy. Electronic address:

Lipopolysaccharides (LPS) are complex amphiphilic macromolecules forming the external leaflet of the outer membrane of Gram-negative bacteria. The LPS glycolipid portion, named Lipid A, is characterized by a disaccharide backbone carrying multiple acyl chains. Some Lipid A bear very-long-chain-fatty-acids (VLCFA), biosynthesized to span the entire lipid membrane profile. The synbiontic Bradyrhizobium BTAi1 strain carries an unique Lipid A specie, named HoLA, in which VLCFA terminus is covalently-bonded to hopanoid, a triterpenoid displaying structural similarity with eukaryotic sterols. Here, we investigate the role of HoLA in regulating self-assembly, microstructure and thermotropicity of lipid membranes composed by 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1'-rac-glycerol. DLS and SANS indicated the formation of multilamellar aggregates whose size increases when the hopanoid is present in the bilayer. EPR and DSC showed that HoLA induced a more rigid and ordered organization among the lipids in the bilayer, evocative of a mechanical strengthening. Notably, the presence of calcium ions promote an increase of the bilayer thickness and vesicles size, leading to low curvature aggregates. These results highlight the key role of the hopanoid covalently-linked to VLCFA in defining the physico-chemical properties of bacterial envelope, thus offering a robust scientific basis for the interpretation of the biological activity of the considered strain.
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http://dx.doi.org/10.1016/j.jcis.2021.03.072DOI Listing
July 2021

Biomolecular Condensates under Extreme Martian Salt Conditions.

J Am Chem Soc 2021 04 23;143(13):5247-5259. Epub 2021 Mar 23.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.
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http://dx.doi.org/10.1021/jacs.1c01832DOI Listing
April 2021

Harnessing Pressure Modulation for Exploring Ligand Binding Reactions in Cosolvent Solutions.

J Phys Chem B 2021 01 11;125(2):539-546. Epub 2021 Jan 11.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227 Dortmund, Germany.

A comprehensive understanding of ligand-protein interactions requires information about all thermodynamic parameters that describe the complexation reaction, and they should be able to provide the necessary information to understand the molecular forces that drive complex formation. Usually, binding studies are performed at ambient pressure conditions. However, in addition to using temperature variation to reveal enthalpic and entropic contributions to ligand binding, complementary pressure-dependent studies providing volumetric properties of the reaction can be beneficial. Changes in partial molar volume can inform about changes in packing and hydration upon ligand binding. Here, after a general discussion of pressure effects on ligand binding reactions, we present a comprehensive study of the effect of pressure and a widely used organic cosolvent, dimethyl sulfoxide (DMSO), on the binding of a small aromatic ligand, proflavine, to the enzyme α-chymotrypsin. We found that DMSO, which acts as a competitive inhibitor for proflavine, has a strong impact on the interaction process, resulting in a decrease of the binding constant. While the reaction performed in neat buffer is basically pressure insensitive, the partial molar volume of the complex in the presence of DMSO is larger compared with the uncomplexed state, rendering the binding constant markedly smaller upon pressurization. We also show that the magnitude and sign of the binding volume provide valuable information about the interaction mechanism and hydration changes, which is of particular interest when cosolvents are present.
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http://dx.doi.org/10.1021/acs.jpcb.0c10212DOI Listing
January 2021

Remodeling of the Conformational Dynamics of Noncanonical DNA Structures by Monomeric and Aggregated α-Synuclein.

J Am Chem Soc 2020 10 19;142(43):18299-18303. Epub 2020 Oct 19.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Street 4a, 44227 Dortmund, Germany.

Research on Parkinson's disease most often focuses on the ability of the protein α-synuclein (α-syn) to form oligomers and amyloid fibrils, and how such species promote brain death. However, there are indications that α-syn also plays a gene-regulatory role in the cell nucleus. Noncanonical tetrahelical nucleic acids, G-quadruplexes (G4Q), and i-motifs have been shown to play an important role in the control of genomic events. Using the conformation-sensitive single-molecule Förster resonance energy transfer technique we show that monomeric and oligomeric α-syn affect G4Qs and i-motifs in a different way and lead to remodeling of their conformational substates. Aggregated α-syn destabilizes the G4Q leading to unfolding. In contrast, both monomeric and aggregated α-syn enhance folding of the i-motif sequence of telomeric DNA. Importantly, macromolecular crowding is able to partially rescue G4Q from unfolding.
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http://dx.doi.org/10.1021/jacs.0c07192DOI Listing
October 2020

Synthesis of new riboflavin modified ODNs: Effect of riboflavin moiety on the G-quadruplex arrangement and stability.

Bioorg Chem 2020 11 1;104:104213. Epub 2020 Sep 1.

Department of Chemical Sciences, University of Napoli Federico II, Complesso di Monte Sant'Angelo, Via Cintia 4, I-80126 Napoli, Italy. Electronic address:

In the panorama of modified G-quadruplexes (G4s) with interesting proprieties, here, it has been reported the synthesis of new modified d(TGGGAG) sequences forming G-quadruplexes, with the insertion of a riboflavin unit (Rf, vitamin B). Exploiting the flavin similarity with the hydrogen bond pattern of guanine and aiming at mimic a typical nucleoside scaffold, the synthesis of the riboflavin building block 3 it has been efficiently carried out. The effect of insertion of riboflavin mimic nucleoside on the G-quadruplex properties has been here, for the first time investigated. A biophysical characterization of Rf-modified sequences (A-D) has been carried out by circular dichroism (CD), fluorescence spectroscopy, differential scanning calorimetry (DSC) and native gel electrophoresis. CD and electrophoresis data have suggested that Rf-modified sequences are able to form parallel tetramolecular G4 structures similar to that of the unmodified sequence. Analysis of the DSC thermograms has revealed that all modified G-quadruplexes have a higher thermal stability compared with the natural sequence, particularly the stabilisation is higher when the Rf residue is introduced at the 3'-end. Further, DSC analysis has revealed that the Rf residues introduced at the 3'-end are able to form additional stabilising interactions, energetically almost comparable to the enthalpic contribution of a G-tetrad. Fluorescence measurement are consistent with this result showing that the Rf residues introduced at 3'-end are able to form stacking interactions with the adjacent bases within the G-quadruplex structure. The whole of data suggested that the introduction of Rf unit can stabilize G-quadruplex structures and can be a promising candidate for future theranostic applications.
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http://dx.doi.org/10.1016/j.bioorg.2020.104213DOI Listing
November 2020

Perturbation of liquid droplets of P-granule protein LAF-1 by the antimicrobial peptide LL-III.

Chem Commun (Camb) 2020 Oct;56(78):11577-11580

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Strasse 4a, D-44227 Dortmund, Germany.

In recent years, liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization. But there is rapidly growing evidence that LLPS may also be associated with a number of medical conditions, including neurodegenerative diseases, by acting as a modulator of pathological protein aggregation. Here we show how LLPS formed by the P-granule protein LAF-1 and RNA can be affected by antimicrobial peptides, such as LL-III, leading to enhanced formation of amorphous protein aggregates and the loss of droplet function as an efficient reaction center and organizational hub.
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http://dx.doi.org/10.1039/d0cc04877aDOI Listing
October 2020

The multifaceted effects of DMSO and high hydrostatic pressure on the kinetic constants of hydrolysis reactions catalyzed by α-chymotrypsin.

Phys Chem Chem Phys 2020 Jul;22(28):16325-16333

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Strasse 4a, D-44227 Dortmund, Germany.

The use of cosolvents and high hydrostatic pressure (HHP) has been described as an efficient means to modulate the stability of enzymes and their catalytic activity. Cosolvents and pressure can lead to increased reaction rates without affecting the stability of the enzyme. Here, we studied the combined effects of one of the most used organic cosolvents, dimethyl sulfoxide (DMSO), and HHP to reveal their combined effect on the kinetic constants of an α-chymotrypsin-catalyzed peptide hydrolysis reaction. The Michaelis constant and the turnover number of the reaction respond differently to the two variables, and we observed an opposite effect of hydrostatic pressure and the dipolar cosolvent DMSO on the kinetic parameters. The results could be rationalized by determining the volume diagram of the reaction at the different solution conditions. In our case, the use of high hydrostatic pressure in concert with DMSO does not lead to an improvement of the enzymatic activity. However, the advantages of DMSO and HHP to increase the temperature stability of the enzyme and to increase the solubility of more hydrophobic substrates could still be useful.
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http://dx.doi.org/10.1039/d0cp03062gDOI Listing
July 2020

Similarities and differences for membranotropic action of three unnatural antimicrobial peptides.

J Pept Sci 2020 Aug 18;26(8):e3270. Epub 2020 Jun 18.

Department of Chemical Sciences, University of Naples Federico II, Naples, Italy.

Previously, we described the design and synthesis of three nine-residue AMPs, P9Nal(SS), P9Trp(SS), and P9Nal(SR), showing high stability in serum and broad spectrum antimicrobial activity. The peptides P9Trp(SS) and P9Nal(SR) differ from P9Nal(SS) for the replacement of the two 2Nal residues with Trp residues and for the replacement of the two Cys (StBu) with Cys (tBu) residues, respectively. These changes led to peptides with a lower hydrophobicity respect to the P9Nal(SS). Interestingly, the three peptides have very similar activity against Gram-negative bacteria. Instead, they exhibit a significant difference towards Gram-positive bacteria, being P9Nal(SS) the most active. In order to evaluate the impact of amino acids substitution on membranotropic activity and rationalize the observed effects in vivo, here, we report the detailed biophysical characterization of the interaction between P9Nal(SR) and P9Trp(SS) and liposomes by combining differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The comparison with the results for the previously characterized P9Nal(SS) peptide reveals similarities and differences on the interaction process and perturbation activities. It was found that the three peptides can penetrate at different extent inside the bilayer upon changing their conformation and inducing lipid domains formation, revealing that the formation of lipid domains is fundamental for the activity against Gram-negative bacteria. On the contrary, the dissimilar activity against Gram-positive bacteria well correlate with the different affinity of peptides for the lipoteichoic acid, a component selectively present in the cell wall of Gram-positive bacteria.
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http://dx.doi.org/10.1002/psc.3270DOI Listing
August 2020

Alteration of Protein Binding Affinities by Aqueous Two-Phase Systems Revealed by Pressure Perturbation.

Sci Rep 2020 05 15;10(1):8074. Epub 2020 May 15.

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Strasse 4a, 44227, Dortmund, Germany.

Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most multifunctional proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo.
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http://dx.doi.org/10.1038/s41598-020-65053-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228918PMC
May 2020

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury.

Sensors (Basel) 2020 Jan 21;20(3). Epub 2020 Jan 21.

Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, Via Salvador Allende, 84081 Baronissi, Italy.

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).
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http://dx.doi.org/10.3390/s20030598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038057PMC
January 2020

Pressure Sensitivity of SynGAP/PSD-95 Condensates as a Model for Postsynaptic Densities and Its Biophysical and Neurological Ramifications.

Chemistry 2020 Aug 13;26(48):11024-11031. Epub 2020 Mar 13.

Physical Chemistry I-Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Strasse 4a, 44227, Dortmund, Germany.

Biomolecular condensates consisting of proteins and nucleic acids can serve critical biological functions, so that some condensates are referred as membraneless organelles. They can also be disease-causing, if their assembly is misregulated. A major physicochemical basis of the formation of biomolecular condensates is liquid-liquid phase separation (LLPS). In general, LLPS depends on environmental variables, such as temperature and hydrostatic pressure. The effects of pressure on the LLPS of a binary SynGAP/PSD-95 protein system mimicking postsynaptic densities, which are protein assemblies underneath the plasma membrane of excitatory synapses, were investigated. Quite unexpectedly, the model system LLPS is much more sensitive to pressure than the folded states of typical globular proteins. Phase-separated droplets of SynGAP/PSD-95 were found to dissolve into a homogeneous solution already at ten-to-hundred bar levels. The pressure sensitivity of SynGAP/PSD-95 is seen here as a consequence of both pressure-dependent multivalent interaction strength and void volume effects. Considering that organisms in the deep sea are under pressures up to about 1 kbar, this implies that deep-sea organisms have to devise means to counteract this high pressure sensitivity of biomolecular condensates to avoid harm. Intriguingly, these findings may shed light on the biophysical underpinning of pressure-related neurological disorders in terrestrial vertebrates.
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http://dx.doi.org/10.1002/chem.201905269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496680PMC
August 2020

Modulation of enzymatic activity by aqueous two-phase systems and pressure - rivalry between kinetic constants.

Chem Commun (Camb) 2020 Jan;56(3):395-398

Physical Chemistry I - Biophysical Chemistry, Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn Strasse 4a, D-44227 Dortmund, Germany.

We studied the combined effects of an aqueous two-phase system (ATPS) invoking liquid-liquid phase separation and pressure on an enzymatic hydrolysis reaction. We show that simple steric crowding effects are not able to explain the kinetic constants and their pressure dependence in the ATPS. Additional contributions, such as changes in water activity and non-specific weak interactions with ATPS components have to be invoked to explain the results obtained. The findings are relevant for understanding cellular processes of piezophiles and might have significant bearings on biotechnological applications using liquid-liquid phase separation and pressure in concert for modulating enzymatic reactions.
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http://dx.doi.org/10.1039/c9cc08065aDOI Listing
January 2020
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