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Molecules (Basel, Switzerland) Jan 2024Multi-step electron transfer reactions are important to the function of many cellular systems. The ways in which such systems have evolved to direct electrons along...
Multi-step electron transfer reactions are important to the function of many cellular systems. The ways in which such systems have evolved to direct electrons along specific pathways are largely understood, but less so are the ways in which the reduction-oxidation potentials of individual redox sites are controlled. We prepared a series of three new artificial variants of azurin where a tyrosine (Tyr109) is situated between the native Cu ion and a Ru(II) photosensitizer tethered to a histidine (His107). Arginine, glutamine, or methionine were introduced as position 122, which is near to Tyr109. We investigated the rate of Cu oxidation by a flash-quench generated Ru(III) oxidant over pH values from 5 to 9. While the identity of the residue at position 122 affects some of the physical properties of Tyr109, the rates of Cu oxidation are only weakly dependent on the identity of the residue at 122. The results highlight that more work is still needed to understand how non-covalent interactions of redox active groups are affected in redox proteins.
Topics: Tyrosine; Electrons; Glutamine; Methionine; Arginine
PubMed: 38257263
DOI: 10.3390/molecules29020350 -
The Journal of Physical Chemistry. B Feb 2024Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such...
Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a β-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the β-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the S-S bonded and S-S nonbonded () forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for azurin compared to that of the form (420 K). As observed in the case of azurin, the unfolding process of the form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual β-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.
Topics: Azurin; Copper; Metalloproteins; Disulfides; Temperature; Ions; Protein Folding
PubMed: 38236012
DOI: 10.1021/acs.jpcb.3c07089 -
Dalton Transactions (Cambridge, England... Jan 2024Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1...
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.
Topics: Azurin; Binding Sites; Copper; Ligands
PubMed: 38170898
DOI: 10.1039/d3dt03372d -
The Journal of Physical Chemistry. B Jan 2024Electron transfer (ET) between neutral and cationic tryptophan residues in the azurin construct [Re(H126)(CO)(dmp)](W124)(W122)Cu (dmp = 4,7-Me-1,10-phenanthroline) was...
Electron transfer (ET) between neutral and cationic tryptophan residues in the azurin construct [Re(H126)(CO)(dmp)](W124)(W122)Cu (dmp = 4,7-Me-1,10-phenanthroline) was investigated by Born-Oppenheimer quantum-mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) simulations. We focused on W124 ← W122 ET, which is the middle step of the photochemical hole-hopping process *Re(CO)(dmp) ← W124 ← W122 ← Cu, where sequential hopping amounts to nearly 10,000-fold acceleration over single-step tunneling (. , , 192-200). In accordance with experiments, UKS-DFT QM/MM/MD simulations identified forward and reverse steps of W124 ↔ W122 ET equilibrium, as well as back ET Re(CO)(dmp) → W124 that restores *Re(CO)(dmp). Strong electronic coupling between the two indoles (≥40 meV in the crossing region) makes the productive W124 ← W122 ET adiabatic. Energies of the two redox states are driven to degeneracy by fluctuations of the electrostatic potential at the two indoles, mainly caused by water solvation, with contributions from the protein dynamics in the W122 vicinity. ET probability depends on the orientation of Re(CO)(dmp) relative to W124 and its rotation diminishes the hopping yield. Comparison with hole hopping in natural systems reveals structural and dynamics factors that are important for designing efficient hole-hopping processes.
Topics: Azurin; Tryptophan; Oxidation-Reduction; Electron Transport; Indoles
PubMed: 38145895
DOI: 10.1021/acs.jpcb.3c06568 -
The Journal of Physical Chemistry... Dec 2023We conducted a theoretical study of electron transport through junctions of the blue-copper azurin from . We found that single-site hopping can lead to either higher or...
We conducted a theoretical study of electron transport through junctions of the blue-copper azurin from . We found that single-site hopping can lead to either higher or lower current values compared to fully coherent transport. This depends on the structural details of the junctions as well as the alignment of the protein orbitals. Moreover, we show how the asymmetry of the curves can be affected by the position of the tip in the junction and that, under specific conditions, such a hopping mechanism is consistent with a fairly low temperature dependence of the current. Finally, we show that increasing the number of hopping sites leads to higher hopping currents. Our findings, from fully quantum calculations, provide deep insight to help guide the interpretation of experimental curves on highly complex systems.
PubMed: 38059566
DOI: 10.1021/acs.jpclett.3c02702 -
Biomacromolecules Jan 2024A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is...
A fusion protein composed of a bacterial protein, azurin, having antineoplastic properties and a thermally responsive structural cationic elastin-like protein (ELP), is designed, cloned, expressed, and purified. A simple method of inverse transition cycle (ITC) is employed to purify the fusion protein azurin-ELP diblock copolymer (d-bc). The molecular weight of the azurin-ELP fusion protein is ∼32 kDa. Further, its self-assembly properties are investigated. Interestingly, the engineered azurin-ELP d-bc in response to increasing temperature shows a dual-step phase separation into biofunctional nanostructures. Around the physiological temperature, azurin-ELP d-bc forms stable coacervates, which is dependent on the concentration and time of incubation. These coacervates are formed below the lower critical solubility temperature (LCST) of the ELP block at physiological temperature. Above LCST, i.e., 50-55°C, micelles of size ranging from 25 to 30 nm are formed. The cytotoxicity of azurin-ELP d-bc depends on the size of the coacervates formed and their cellular uptake at physiological temperature. Further, MTT assay of azurin-ELP d-bc in the cross-linked micelles prepared ex situ shows > six times higher killing of LNCaP cells than the unimeric form of azurin-ELP at 5 μM concentration. The flow cytometric results of these micelles at 20 μM concentration show ∼97% LNCaP cells in the apoptotic phase. Thus, azurin-ELP cross-linked micelles have enhanced potential for anticancer therapy due to their higher avidity.
Topics: Humans; Male; Elastin-Like Polypeptides; Micelles; Azurin; Peptides; Elastin; Prostatic Neoplasms
PubMed: 38047916
DOI: 10.1021/acs.biomac.3c01125 -
International Journal of Biological... Jan 2024Chemical protein (semi-)synthesis is a powerful technique allowing the incorporation of unnatural functionalities at any desired protein site. Herein we describe a...
Chemical protein (semi-)synthesis is a powerful technique allowing the incorporation of unnatural functionalities at any desired protein site. Herein we describe a facile one-pot semi-synthetic strategy for the construction of a type 2 copper center in the active site of azurin, which is achieved by substitution of Met121 with unnatural amino acid residues bearing a strong ligand N,N-bis(pyridylmethyl)amine (DPA) to mimic the function of typical histidine brace-bearing copper monooxygenases, such as lytic polysaccharide monooxygenases (LPMOs) involved in polysaccharide breakdown. The semi-synthetic proteins were routinely obtained in over 10-mg scales to allow for spectroscopic measurements (UV-Vis, CD, and EPR), which provides structural evidences for the Cu-DPA-modified azurins. 4-nitrophenyl-β-D-glucopyranoside (PNPG) was used as a model substrate for the HO-driven oxidative cleavage reaction facilitated by semi-synthetic azurins, and the Cu-6 complex showed a highest activity (TTN 253). Interestingly, our semi-synthetic azurins were able to tolerate high HO concentrations (up to 4000-fold of the enzyme), making them promising for practical applications. Collectively, we establish that chemical protein synthesis can be exploited as a reliable technology in affording large quantities of artificial metalloproteins to facilitate the transformation of challenging chemical reactions.
Topics: Azurin; Copper; Hydrogen Peroxide; Mixed Function Oxygenases; Oxidative Stress; Polysaccharides
PubMed: 38000595
DOI: 10.1016/j.ijbiomac.2023.128083 -
Journal of Biological Inorganic... Dec 2023Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but...
Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn-CP protein was much slower than that of the Zn-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.
Topics: Azurin; Protein Folding; Catalytic Domain; Apoproteins; Metals; Circular Dichroism; Kinetics
PubMed: 37957357
DOI: 10.1007/s00775-023-02023-z -
Angewandte Chemie (International Ed. in... Dec 2023The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu reduction potential (E°' ) to match those of its redox partners, enabling efficient electron...
Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations.
The key to type 1 copper (T1Cu) function lies in the fine tuning of the Cu reduction potential (E°' ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°' in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°' of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°' . While minimal and small E°' increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°' increase. Moreover, the influence of these mutations on E°' is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°' of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (k /K ) relative to WT-SLAC.
Topics: Copper; Laccase; Mutation; Oxidation-Reduction; Streptomyces coelicolor
PubMed: 37926680
DOI: 10.1002/anie.202314019 -
Molecular Pharmaceutics Dec 2023Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a...
Erythropoietin-producing hepatocellular (Eph) receptors and their ligands, ephrins, are the largest subfamily of receptor tyrosine kinases (RTKs) that have emerged as a new class of cancer biomarkers due to their aberrant expression in cancer progression. The activation of Eph receptors either due to their hyperexpression or via high affinity binding with their respective ephrin ligands initiates a cascade of signals that impacts cancer development and progression. In prostate cancer, the overexpression of the EphA6 receptor has been correlated with increased metastatic potential. Azurin, a small redox protein, is known to prevent tumor progression by binding to cell surface Eph receptors, inhibiting its autophosphorylation in the kinase domain and thereby disrupting Eph-ephrin signaling. Hence, a self-assembled, theranostic nanosystem of recombinant fusion protein hisEGFP-azu (80-128) was designed by conjugating enhanced green fluorescent protein (EGFP) with the C-terminal region of azurin. This design was inspired by the binding study, where the analogue of ephrinA, hisEGFP-azu (80-128) showed higher binding affinity for the EphA6 receptor than the ephrinA ligands. The hisEGFP-azu (80-128) nanosystem which assembled as nanoparticles was tested for its ability to simultaneously detect and kill the prostate cancer cells, LNCaP. This was achieved by specifically targeting EphA6 receptors overexpressed on the cancer cell surface via C-terminal peptide, azu (80-128). Herein, we report antiproliferative, apoptotic, antimigratory, and anti-invasive effects of this nanosystem on LNCaP cells, while having no similar effects on EphA6 negative human normal lung cells, WI-38.
Topics: Male; Humans; Receptors, Eph Family; Azurin; Receptor, EphA6; Precision Medicine; Prostatic Neoplasms; Ephrins
PubMed: 37906960
DOI: 10.1021/acs.molpharmaceut.3c00387