Joanna Makowska - Academia.edu (original) (raw)
Papers by Joanna Makowska
Journal of Molecular Liquids, Nov 1, 2021
Abstract Understanding the metal ion-protein interactions is crucial in transport, accumulation, ... more Abstract Understanding the metal ion-protein interactions is crucial in transport, accumulation, and excretion of metals. Furthermore, it seems to be highly important to investigate the role of such complexes in many diseases, such as cancer and neurodegenerative disorders. Unexpectedly, such studies in the systems comprising peptides and Ni2+ ions are still scarce and should be given more importance – particularly considering the toxicity and carcinogenicity of nickel compounds on the living organisms. We report herein the results of a study on the interactions of Ni2+ ions and four pentapeptide analogs: EYHHQ (p1), EHYHQ (p2), HEYHQ (p3), and HEYQH (p4). Potentiometric titration was used to study acid-base properties as well as binding properties of the investigated peptides. Furthermore, stoichiometry of the peptide complexes with Ni2+ ions was evaluated by steady-state fluorescence spectroscopy and potentiometric titration method. Additionally, based on the density functional theory (DFT) calculations, we propose the most likely structures of the complexes of the peptides p1-p4 with Ni2+ ions. It was found that all the peptides form 1:1 and 1:2 thermodynamically stable complexes with Ni2+ cation. In particular, we observed that the interaction between the metal ion and histidine-containing peptide sequences does not depend only on histidines splicing, but also on their orientation in respect to other amino acids in the sequence. Also, the closer proximity of His residue in these short peptides does not increase complex stability, due to stacking interaction between the neighboring imidazole rings.
Journal of Physical Chemistry B, Feb 27, 2007
Simple analytical functions consisting of electrostatic, polarization, Lennard-Jones, and cavity ... more Simple analytical functions consisting of electrostatic, polarization, Lennard-Jones, and cavity terms were proposed to express the potential of mean force of particles interacting in water analytically. A simple analytical approximation based on the integral over the surface-hydration energy density of the interacting sites was derived for the cavity term; the solvation energy density of each site is represented as a difference of two Gaussian terms. This expression can easily be generalized to interaction sites with non-spherical symmetry. The analytical expressions were fitted to the potential of mean force of model systems determined by umbrella-sampling molecular dynamics. The analytical formulas fitted the potentials of mean force very well, and the resulting parameters of the expressions were physically reasonable.
Journal of Back and Musculoskeletal Rehabilitation, Jan 29, 2007
Journal of Molecular Structure-theochem, Mar 1, 2005
In order to determine the preferred protonation and deprotonation sites for four isomers of methy... more In order to determine the preferred protonation and deprotonation sites for four isomers of methyl 3-amino-2,3-dideoxyhexopyranoside with the α/β-d-arabino and β/α-d-ribo configurations, ab initio quantum chemical calculations were performed in the gaseous phase with two methods: Restricted Hartree Fock (RHF) and Møller-Plesset (MP2), as well as after consideration of the solvation effects within the Polarizable Continuum Model (PCM). The energy
Journal of Molecular Liquids, Oct 1, 2021
Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine ... more Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine serum albumin (BSA) a set of experimental methods, namely isothermal titration calorimetry, conductometric titration, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy, supported by in silico analysis have been applied. The influence of pH and temperature on the binding mode has been revealed. At a low molar ratio of SDS to BSA up to ca. 16:1, there are at least two structurally distinct binding sites in BSA. The formation of SDS-BSA complexes is an enthalpy-driven process in which the van der Waals interactions play a crucial role. The first binding site, located close to the Trp-134 residue within the sub-domain IA, is pH-independent and binds two molecules of SDS per one molecule of BSA whereas the total number of SDS molecules bound to the second site of albumin is affected by temperature and pH. The saturation of the first binding site of BSA (ca. 0.009 mg of SDS per 1 mg of BSA) is sufficient to thermally stabilize the helical conformation of BSA. The presented results have important structural and thermodynamic implications to understand the influence of a widely used anionic surfactant on globular protein functionality in modern branches of chemistry.
Microbiology spectrum, Oct 26, 2022
Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public healt... more Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens.
Journal of Physical Chemistry B, Jan 13, 2022
One of the definitions of hydrophobic interactions is the aggregation of nonpolar particles in a ... more One of the definitions of hydrophobic interactions is the aggregation of nonpolar particles in a polar solvent, such as water. While this phenomenon appears to be very simple, it is crucial for many complex processes, such as protein folding, to take place. In this work, the hydrophobic association of adamantane and hexane at various temperatures and ionic strengths was studied using molecular dynamics simulations with the AMBER 16.0 program and the GAFF force field. The potentials of mean force of hydrophobic dimer formation, as well as the excess free energy, excess energy, excess entropy, and excess heat capacity corresponding to the formation of the contact minimum, were determined and analyzed. For both systems, the depth of the contact minimum in the potential of mean force was found to increase with both temperature and ionic strength. The excess heat capacity of the association at the contact minimum and T = 298 K was found to be negative and to decrease, while the excess entropy and energy were found to be positive and to increase for both systems, the changes being more pronounced for the hexane dimer. The excess heat capacity is also greater in absolute value for the hexane dimer.
![Research paper thumbnail of Metal-Ion Interactions with Dodecapeptide Fragments of Human Cationic Antimicrobial Protein LL-37 [hCAP(134–170)]](https://attachments.academia-assets.com/115598536/thumbnails/1.jpg)
](The Journal of Physical Chemistry B
Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were... more Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were used to determine potential metal binding sites in human cationic antimicrobial protein (hCAP) corresponding to overlapping the dodecapeptide sequences of hCAP(134−170) referred to as LL-37. The correct antibacterial action of LL-37 is closely related to its established unique structure. Disturbances in the LL-37 structure (e.g., unwanted presence of metal ions) lead to a radical change in its biological functions. Five fragments of the LL-37 [hCAP(134−170)], namely, hCAP(134−145) (A1), hCAP(140−151) (A2), hCAP(146−157) (A3), hCAP(152−163) (A4), and hCAP(159−170) (A5), were taken into account and their affinity to Mn(II) and Zn(II) ions was rigorously assessed. We prove that only three of the investigated peptides (A1, A2, and A5) are capable of forming thermodynamically stable complexes with metal ions. Additionally, based on density functional theory (DFT) calculations, we propose the most likely coordination modes of metal(II) to peptides as well as discuss the chemical nature of the interactions. Finally, we present the structural features of the strongest binding peptide, hCAP(159−170), responsible for the metal binding. The presented results provide important structural and thermodynamic information to understand the influence of some metal ions on the activity of hCAP(134−170).
Journal of Molecular Liquids, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Special Issue Conference Abstract Book CNS 2018, 2018
The Journal of Chemical Thermodynamics, 2019
Isothermal titration calorimetry (ITC) and potentiometric titration (PT) methods were used to stu... more Isothermal titration calorimetry (ITC) and potentiometric titration (PT) methods were used to study the interactions of copper(II) ions with GAG, GDG, GKG, and GHG peptides. The calorimetric measurements were run in a MES buffer with the pH value of 6.0 at 298.15 K. Based on the results of PT data supported by theoretical calculations, the dissociation constants were calculated for the investigated peptides. The quantification of the proton competition with the metal for the peptide and incorporation it into the ITC data analysis enabled to obtain the pH-independent parameters, namely the binding constants (K) and the free energy of binding (DG). Furthermore, the relationship between the proposed coordination modes of the considered peptides and the thermodynamic parameters (K, DG and DH) has been discussed.
Symmetry, 2020
In the following paper, we present the results of our studies on the interactions of the Aβ1-42 p... more In the following paper, we present the results of our studies on the interactions of the Aβ1-42 peptide and its three short fragments, namely Aβ5-16 (RHDSGYEVHHQK; HZ1), Aβ8-13 (SGYEVH; HZ2), and Aβ8-12 (SGYEV; HZ3) with selected painkillers (ibuprofen and aspirin) and compounds of natural origin (anabasine and epinephrine). Steady-state fluorescence spectroscopy was used to study the binding properties of the selected systems. Additionally, based on molecular dynamics (MD) calculations supported by NMR-derived restrains, we have proposed the most likely area of the interactions of Aβ1-42 and Aβ5-16 peptides with the investigated compounds. The influence of symmetrically oriented side chains of amino acid residues present in the first part of the Aβ1-42 sequence on the stability of the resulting complexes has been discussed. Finally, the changes in the peptide structures on account of complex formation were analyzed.
DOAJ (DOAJ: Directory of Open Access Journals), Oct 1, 2014
We examined the effect of like-charged residues on the conformation of an original nine amino-aci... more We examined the effect of like-charged residues on the conformation of an original nine amino-acid-residue fragment of the human Pin1 WW domain (hPin1) with the following sequence: Ac-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-NH 2 (U9). This was facilitated by CD and NMR spectroscopic measurements, and molecular dynamics calculations. Our earlier studies suggested that the presence of like-charged residues at the end of a short polypeptide chain composed of nonpolar residues could induce a chain reversal. For the U9 peptide, canonical MD simulations with NMR-derived restraints demonstrated the presence of ensembles of structures with a tendency to form a β-chain reversal. Additionally, thermal stabilities of the peptide under study were measured using differential scanning calorimetry (DSC). The estimated well defined phase transition point showed that conformational equilibria in the U9 peptide were strongly dependent on temperature.
International Journal of Molecular Sciences
We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus the... more We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysi...
Recent Progress in Computational Sciences and Engineering, 2019
Molecules, 2021
The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) ha... more The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]− for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]− ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = −9.67 ± 0.14 kcal mol−1; ΔH(ITC)1 = −3.14 ± 0.12 kcal mol−1; TΔS(ITC)1 = −6.53 kcal mol−1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 ...
Journal of Molecular Liquids, 2021
Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine ... more Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine serum albumin (BSA) a set of experimental methods, namely isothermal titration calorimetry, conductometric titration, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy, supported by in silico analysis have been applied. The influence of pH and temperature on the binding mode has been revealed. At a low molar ratio of SDS to BSA up to ca. 16:1, there are at least two structurally distinct binding sites in BSA. The formation of SDS-BSA complexes is an enthalpy-driven process in which the van der Waals interactions play a crucial role. The first binding site, located close to the Trp-134 residue within the sub-domain IA, is pH-independent and binds two molecules of SDS per one molecule of BSA whereas the total number of SDS molecules bound to the second site of albumin is affected by temperature and pH. The saturation of the first binding site of BSA (ca. 0.009 mg of SDS per 1 mg of BSA) is sufficient to thermally stabilize the helical conformation of BSA. The presented results have important structural and thermodynamic implications to understand the influence of a widely used anionic surfactant on globular protein functionality in modern branches of chemistry.
Journal of Solution Chemistry, 2018
Isothermal titration calorimetry, potentiometric titration and circular dichroism spectroscopy we... more Isothermal titration calorimetry, potentiometric titration and circular dichroism spectroscopy were used to study the interaction of copper(II) ions with Argireline (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH 2) and three of its point mutation derivatives:
Journal of Molecular Liquids, Nov 1, 2021
Abstract Understanding the metal ion-protein interactions is crucial in transport, accumulation, ... more Abstract Understanding the metal ion-protein interactions is crucial in transport, accumulation, and excretion of metals. Furthermore, it seems to be highly important to investigate the role of such complexes in many diseases, such as cancer and neurodegenerative disorders. Unexpectedly, such studies in the systems comprising peptides and Ni2+ ions are still scarce and should be given more importance – particularly considering the toxicity and carcinogenicity of nickel compounds on the living organisms. We report herein the results of a study on the interactions of Ni2+ ions and four pentapeptide analogs: EYHHQ (p1), EHYHQ (p2), HEYHQ (p3), and HEYQH (p4). Potentiometric titration was used to study acid-base properties as well as binding properties of the investigated peptides. Furthermore, stoichiometry of the peptide complexes with Ni2+ ions was evaluated by steady-state fluorescence spectroscopy and potentiometric titration method. Additionally, based on the density functional theory (DFT) calculations, we propose the most likely structures of the complexes of the peptides p1-p4 with Ni2+ ions. It was found that all the peptides form 1:1 and 1:2 thermodynamically stable complexes with Ni2+ cation. In particular, we observed that the interaction between the metal ion and histidine-containing peptide sequences does not depend only on histidines splicing, but also on their orientation in respect to other amino acids in the sequence. Also, the closer proximity of His residue in these short peptides does not increase complex stability, due to stacking interaction between the neighboring imidazole rings.
Journal of Physical Chemistry B, Feb 27, 2007
Simple analytical functions consisting of electrostatic, polarization, Lennard-Jones, and cavity ... more Simple analytical functions consisting of electrostatic, polarization, Lennard-Jones, and cavity terms were proposed to express the potential of mean force of particles interacting in water analytically. A simple analytical approximation based on the integral over the surface-hydration energy density of the interacting sites was derived for the cavity term; the solvation energy density of each site is represented as a difference of two Gaussian terms. This expression can easily be generalized to interaction sites with non-spherical symmetry. The analytical expressions were fitted to the potential of mean force of model systems determined by umbrella-sampling molecular dynamics. The analytical formulas fitted the potentials of mean force very well, and the resulting parameters of the expressions were physically reasonable.
Journal of Back and Musculoskeletal Rehabilitation, Jan 29, 2007
Journal of Molecular Structure-theochem, Mar 1, 2005
In order to determine the preferred protonation and deprotonation sites for four isomers of methy... more In order to determine the preferred protonation and deprotonation sites for four isomers of methyl 3-amino-2,3-dideoxyhexopyranoside with the α/β-d-arabino and β/α-d-ribo configurations, ab initio quantum chemical calculations were performed in the gaseous phase with two methods: Restricted Hartree Fock (RHF) and Møller-Plesset (MP2), as well as after consideration of the solvation effects within the Polarizable Continuum Model (PCM). The energy
Journal of Molecular Liquids, Oct 1, 2021
Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine ... more Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine serum albumin (BSA) a set of experimental methods, namely isothermal titration calorimetry, conductometric titration, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy, supported by in silico analysis have been applied. The influence of pH and temperature on the binding mode has been revealed. At a low molar ratio of SDS to BSA up to ca. 16:1, there are at least two structurally distinct binding sites in BSA. The formation of SDS-BSA complexes is an enthalpy-driven process in which the van der Waals interactions play a crucial role. The first binding site, located close to the Trp-134 residue within the sub-domain IA, is pH-independent and binds two molecules of SDS per one molecule of BSA whereas the total number of SDS molecules bound to the second site of albumin is affected by temperature and pH. The saturation of the first binding site of BSA (ca. 0.009 mg of SDS per 1 mg of BSA) is sufficient to thermally stabilize the helical conformation of BSA. The presented results have important structural and thermodynamic implications to understand the influence of a widely used anionic surfactant on globular protein functionality in modern branches of chemistry.
Microbiology spectrum, Oct 26, 2022
Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public healt... more Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens.
Journal of Physical Chemistry B, Jan 13, 2022
One of the definitions of hydrophobic interactions is the aggregation of nonpolar particles in a ... more One of the definitions of hydrophobic interactions is the aggregation of nonpolar particles in a polar solvent, such as water. While this phenomenon appears to be very simple, it is crucial for many complex processes, such as protein folding, to take place. In this work, the hydrophobic association of adamantane and hexane at various temperatures and ionic strengths was studied using molecular dynamics simulations with the AMBER 16.0 program and the GAFF force field. The potentials of mean force of hydrophobic dimer formation, as well as the excess free energy, excess energy, excess entropy, and excess heat capacity corresponding to the formation of the contact minimum, were determined and analyzed. For both systems, the depth of the contact minimum in the potential of mean force was found to increase with both temperature and ionic strength. The excess heat capacity of the association at the contact minimum and T = 298 K was found to be negative and to decrease, while the excess entropy and energy were found to be positive and to increase for both systems, the changes being more pronounced for the hexane dimer. The excess heat capacity is also greater in absolute value for the hexane dimer.
The Journal of Physical Chemistry B
Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were... more Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were used to determine potential metal binding sites in human cationic antimicrobial protein (hCAP) corresponding to overlapping the dodecapeptide sequences of hCAP(134−170) referred to as LL-37. The correct antibacterial action of LL-37 is closely related to its established unique structure. Disturbances in the LL-37 structure (e.g., unwanted presence of metal ions) lead to a radical change in its biological functions. Five fragments of the LL-37 [hCAP(134−170)], namely, hCAP(134−145) (A1), hCAP(140−151) (A2), hCAP(146−157) (A3), hCAP(152−163) (A4), and hCAP(159−170) (A5), were taken into account and their affinity to Mn(II) and Zn(II) ions was rigorously assessed. We prove that only three of the investigated peptides (A1, A2, and A5) are capable of forming thermodynamically stable complexes with metal ions. Additionally, based on density functional theory (DFT) calculations, we propose the most likely coordination modes of metal(II) to peptides as well as discuss the chemical nature of the interactions. Finally, we present the structural features of the strongest binding peptide, hCAP(159−170), responsible for the metal binding. The presented results provide important structural and thermodynamic information to understand the influence of some metal ions on the activity of hCAP(134−170).
Journal of Molecular Liquids, 2020
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Special Issue Conference Abstract Book CNS 2018, 2018
The Journal of Chemical Thermodynamics, 2019
Isothermal titration calorimetry (ITC) and potentiometric titration (PT) methods were used to stu... more Isothermal titration calorimetry (ITC) and potentiometric titration (PT) methods were used to study the interactions of copper(II) ions with GAG, GDG, GKG, and GHG peptides. The calorimetric measurements were run in a MES buffer with the pH value of 6.0 at 298.15 K. Based on the results of PT data supported by theoretical calculations, the dissociation constants were calculated for the investigated peptides. The quantification of the proton competition with the metal for the peptide and incorporation it into the ITC data analysis enabled to obtain the pH-independent parameters, namely the binding constants (K) and the free energy of binding (DG). Furthermore, the relationship between the proposed coordination modes of the considered peptides and the thermodynamic parameters (K, DG and DH) has been discussed.
Symmetry, 2020
In the following paper, we present the results of our studies on the interactions of the Aβ1-42 p... more In the following paper, we present the results of our studies on the interactions of the Aβ1-42 peptide and its three short fragments, namely Aβ5-16 (RHDSGYEVHHQK; HZ1), Aβ8-13 (SGYEVH; HZ2), and Aβ8-12 (SGYEV; HZ3) with selected painkillers (ibuprofen and aspirin) and compounds of natural origin (anabasine and epinephrine). Steady-state fluorescence spectroscopy was used to study the binding properties of the selected systems. Additionally, based on molecular dynamics (MD) calculations supported by NMR-derived restrains, we have proposed the most likely area of the interactions of Aβ1-42 and Aβ5-16 peptides with the investigated compounds. The influence of symmetrically oriented side chains of amino acid residues present in the first part of the Aβ1-42 sequence on the stability of the resulting complexes has been discussed. Finally, the changes in the peptide structures on account of complex formation were analyzed.
DOAJ (DOAJ: Directory of Open Access Journals), Oct 1, 2014
We examined the effect of like-charged residues on the conformation of an original nine amino-aci... more We examined the effect of like-charged residues on the conformation of an original nine amino-acid-residue fragment of the human Pin1 WW domain (hPin1) with the following sequence: Ac-Arg-Met-Ser-Arg-Ser-Ser-Gly-Arg-Val-NH 2 (U9). This was facilitated by CD and NMR spectroscopic measurements, and molecular dynamics calculations. Our earlier studies suggested that the presence of like-charged residues at the end of a short polypeptide chain composed of nonpolar residues could induce a chain reversal. For the U9 peptide, canonical MD simulations with NMR-derived restraints demonstrated the presence of ensembles of structures with a tendency to form a β-chain reversal. Additionally, thermal stabilities of the peptide under study were measured using differential scanning calorimetry (DSC). The estimated well defined phase transition point showed that conformational equilibria in the U9 peptide were strongly dependent on temperature.
International Journal of Molecular Sciences
We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus the... more We present a structural and functional analysis of the DNA polymerase of thermophilic Thermus thermophilus MAT72 phage vB_Tt72. The enzyme shows low sequence identity (<30%) to the members of the type-A family of DNA polymerases, except for two yet uncharacterized DNA polymerases of T. thermophilus phages: φYS40 (91%) and φTMA (90%). The Tt72 polA gene does not complement the Escherichia colipolA− mutant in replicating polA-dependent plasmid replicons. It encodes a 703-aa protein with a predicted molecular weight of 80,490 and an isoelectric point of 5.49. The enzyme contains a nucleotidyltransferase domain and a 3′-5′ exonuclease domain that is engaged in proofreading. Recombinant enzyme with His-tag at the N-terminus was overproduced in E. coli, subsequently purified by immobilized metal affinity chromatography, and biochemically characterized. The enzyme exists in solution in monomeric form and shows optimum activity at pH 8.5, 25 mM KCl, and 0.5 mM Mg2+. Site-directed analysi...
Recent Progress in Computational Sciences and Engineering, 2019
Molecules, 2021
The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) ha... more The binding interactions of bovine serum albumin (BSA) with tetraphenylborate ions ([B(Ph)4]−) have been investigated by a set of experimental methods (isothermal titration calorimetry, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy) and molecular dynamics-based computational approaches. Two sets of structurally distinctive binding sites in BSA were found under the experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K). The obtained results, supported by the competitive interactions experiments of SDS with [B(Ph)4]− for BSA, enabled us to find the potential binding sites in BSA. The first site is located in the subdomain I A of the protein and binds two [B(Ph)4]− ions (logK(ITC)1 = 7.09 ± 0.10; ΔG(ITC)1 = −9.67 ± 0.14 kcal mol−1; ΔH(ITC)1 = −3.14 ± 0.12 kcal mol−1; TΔS(ITC)1 = −6.53 kcal mol−1), whereas the second site is localized in the subdomain III A and binds five ions (logK(ITC)2 = 5.39 ± 0.06; ΔG(ITC)2 ...
Journal of Molecular Liquids, 2021
Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine ... more Abstract To rigorously characterize the interactions of sodium dodecyl sulfate (SDS) with bovine serum albumin (BSA) a set of experimental methods, namely isothermal titration calorimetry, conductometric titration, steady-state fluorescence spectroscopy, differential scanning calorimetry and circular dichroism spectroscopy, supported by in silico analysis have been applied. The influence of pH and temperature on the binding mode has been revealed. At a low molar ratio of SDS to BSA up to ca. 16:1, there are at least two structurally distinct binding sites in BSA. The formation of SDS-BSA complexes is an enthalpy-driven process in which the van der Waals interactions play a crucial role. The first binding site, located close to the Trp-134 residue within the sub-domain IA, is pH-independent and binds two molecules of SDS per one molecule of BSA whereas the total number of SDS molecules bound to the second site of albumin is affected by temperature and pH. The saturation of the first binding site of BSA (ca. 0.009 mg of SDS per 1 mg of BSA) is sufficient to thermally stabilize the helical conformation of BSA. The presented results have important structural and thermodynamic implications to understand the influence of a widely used anionic surfactant on globular protein functionality in modern branches of chemistry.
Journal of Solution Chemistry, 2018
Isothermal titration calorimetry, potentiometric titration and circular dichroism spectroscopy we... more Isothermal titration calorimetry, potentiometric titration and circular dichroism spectroscopy were used to study the interaction of copper(II) ions with Argireline (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH 2) and three of its point mutation derivatives: