Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer.

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

Investigation of the Interaction between Mechanosynthesized ZnS Nanoparticles and Albumin Using Fluorescence Spectroscopy.

In this paper, ZnS nanoparticles were bioconjugated with bovine serum albumin and prepared in a form of nanosuspension using a wet circulation grinding. The stable nanosuspension with monomodal particle size distribution (d50 = 137 nm) and negative zeta potential (-18.3 mV) was obtained. The sorption kinetics and isotherm were determined. Interactions between ZnS and albumin were studied using the fluorescence techniques. The quenching mechanism, describing both static and dynamic interactions, was investigated. Various parameters were calculated, including the quenching rate constant, binding constant, stoichiometry of the binding process, and accessibility of fluorophore to the quencher. It has been found that tryptophan, in comparison to tyrosine, can be closer to the binding site established by analyzing the synchronous fluorescence spectra. The cellular mechanism in multiple myeloma cells treated with nanosuspension was evaluated by fluorescence assays for quantification of apoptosis, assessment of mitochondrial membrane potential and evaluation of cell cycle changes. The preliminary results confirm that the nontoxic nature of ZnS nanoparticles is potentially applicable in drug delivery systems. Additionally, slight changes in the secondary structure of albumin, accompanied by a decrease in α-helix content, were investigated using the FTIR method after analyzing the deconvoluted Amide I band spectra of ZnS nanoparticles conjugated with albumin. Thermogravimetric analysis and long-term stability studies were also performed to obtain a complete picture about the studied system.

Nano-bio interface between As4S4 nanoparticles and albumin influenced by wet stirred media milling.

Arsenic sulfide (As4S4) nanoparticles have been intensively researched as a promising drug in a cancer treatment. For the first time, the interaction between As4S4 and bovine serum albumin has been studied in this paper. Initially, the sorption kinetics of albumin on the surface of nanoparticles was investigated. Subsequently, its structural changes influenced by interaction with the As4S4 nanoparticles during wet stirred media milling were studied in deep. Both the dynamic and static quenching were detected after analyzing the fluorescence quenching spectra. From the synchronous fluorescence spectra it was investigated, that the fluorescence intensity for tyrosine residues decreased by about 55%, and for tryptophan it was about 80%. It indicates the fluorescence from tryptophan is more intense and gets more efficiently quenched than those from tyrosine residues in presence of As4S4, implying that the tryptophan can be closer to the binding site. From the circular dichroisms and FTIR spectra it was observed that conformation of the protein remains almost unchanged. The content of appropriate secondary structures was determined by deconvolution of the absorption peak attributed to the amide I band in FTIR spectra. The preliminary anti-tumor cytotoxic effect of prepared albumin-As4S4 system was also tested on multiple myeloma cell lines.

Anion-Specific Effects on the Alkaline State of Cytochrome c.

Specific effects of anions on the structure, thermal stability, and peroxidase activity of native (state III) and alkaline (state IV) cytochrome c (cyt c) have been studied by the UV-VIS absorbance spectroscopy, intrinsic tryptophan fluorescence, and circular dichroism. Thermal and isothermal denaturation monitored by the tryptophan fluorescence and circular dichroism, respectively, implied lower stability of cyt c state IV in comparison with the state III. The pKa value of alkaline isomerization of cyt c depended on the present salts, i.e., kosmotropic anions increased and chaotropic anions decreased pKa (Hofmeister effect on protein stability). The peroxidase activity of cyt c in the state III, measured by oxidation of guaiacol, showed clear dependence on the salt position in the Hofmeister series, while cyt c in the alkaline state lacked the peroxidase activity regardless of the type of anions present in the solution. The alkaline isomerization of cyt c in the presence of 8 M urea, measured by Trp59 fluorescence, implied an existence of a high-affinity non-native ligand for the heme iron even in a partially denatured protein conformation. The conformation of the cyt c alkaline state in 8 M urea was considerably modulated by the specific effect of anions. Based on the Trp59 fluorescence quenching upon titration to alkaline pH in 8 M urea and molecular dynamics simulation, we hypothesize that the Lys79 conformer is most likely the predominant alkaline conformer of cyt c. The high affinity of the sixth ligand for the heme iron is likely a reason of the lack of peroxidase activity of cyt c in the alkaline state.

Conformational properties of LOV2 domain and its C450A variant within broad pH region.

LOV2 (Light-Oxygen-Voltage) domain from Avena sativa phototropin 1 (AsLOV2) belongs to the superfamily of PAS (Per-Arnt-Sim) domains, members of which function as signaling sensors. AsLOV2 undergoes a conformational change upon blue-light absorption by its FMN cofactor. AsLOV2 wild type (wt) is intensively studied as a photo-switchable element in conjugation with various proteins. On the other hand, its variant AsLOV2 with replaced cysteinyl residue C450, which is critical for the forming a covalent adduct with FMN upon irradiation, forms a precursor for some recently developed genetically encoded photosensitizers. In the presented work, we investigated conformational properties of AsLOV2 wt and its variant C450A by circular dichroism, tryptophan and FMN fluorescence, and differential scanning calorimetry in dependence on pH and temperature. We show that both variants are similarly sensitive towards pH of solvent. On the other hand, the mutation C450A leads to a more stable AsLOV2 variant in comparison with the wild type. Thermal transitions of the AsLOV2 proteins monitored by circular dichroism indicate the presence of significant residual structure in thermally-denatured states of both proteins in the pH range from 4 to 9. Both pH- and thermal- transitions of AsLOV2 are accompanied by FMN leaching to solvent. Higher stability, reversibility of thermal transitions, and efficiency of FMN rebinding in the case of C450A variant suggest that the cofactor release may be modulated by suitable mutations in combination with a suitable physicochemical perturbation. These findings can have implications for a design of genetically encoded photosensitizers.

Hofmeister effect on catalytic properties of chymotrypsin is substrate-dependent.

Effect of Hofmeister sodium salts, sulfate, chloride, bromide and perchlorate, on catalytic properties and stability of chymotrypsin has been studied by absorbance and circular dichroism spectroscopies. To address Hofmeister effect on activity of chymotrypsin, two different substrates, N-benzoyl-L-tyrosine ethyl ester and amide N-succinyl-L-phenylalanine-p-nitroanilide, were used. Catalytic activity of chymotrypsin is dependent on salt concentration and position of anion in Hofmeister series. The enzyme activity for both substrates is only slightly affected by chaotropic anions and increases with kosmotropic nature of anions. While the trend of Hofmeister effect on chymotrypsin catalysis is similar for both substrates, the amplitude of the effect significantly differs. In the presence of 1 M sulfate, catalytic efficiency increased by ~2-fold for the ester but ~20-fold for the amide substrate. Positive correlation between stability and activity of chymotrypsin indicates the interdependence of these enzyme properties and is in agreement with recently developed macromolecular rate theory suggesting an important role of protein dynamics in enzyme catalysis. Linear dependencies of catalytic properties of chymotrypsin with partitioning of anions at bulk water/air as well as at hydrocarbon surface strongly indicate that the modulated enzyme properties are results of direct interaction of anions with protein surface.

Peroxidase activity of cytochrome c in its compact state depends on dynamics of the heme region.

Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0-4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions.

Correlation of lysozyme activity and stability in the presence of Hofmeister series anions.

Enzymatic activity and stability of lysozyme in the presence of salts have been studied by fluorescence spectroscopy and differential scanning calorimetry, respectively. The effect of sodium salts of sulfate, acetate, chloride, bromide, thiocyanate, and perchlorate on lysozyme properties depends on anion concentration as well as on position of anion in the Hofmeister series. Kosmotropic anions (sulfate and acetate) increase stability and activate the enzyme while chaotropic anions (bromide, thiocyanate and perchlorate) including chloride decrease stability and inhibits the enzyme activity. Strong correlation between stability and activity of lysozyme suggest the interdependence of these enzyme properties in the presence of salts. The fact that the properties of lysozyme correlate with partition coefficients of anions at hydrocarbon surface clearly indicates that Hofmeister effect of anions is mediated by their interactions with nonpolar parts of the enzyme surface despite its high positive net charge at studied conditions. The efficiency of the anions in affecting both activity and stability of lysozyme also correlates with other anion-related parameters most notably with polarizability of monovalent anions. The presented work points to a critical role of interaction of anions with nonpolar protein surface for the Hofmeister effect. Moreover, the simultaneous investigation of protein stability and activity, in the relation with the Hofmeister effect, provides important information regarding stability/rigidity of enzyme structure for its catalytic activity.

Delipidation of cytochrome c oxidase from Rhodobacter sphaeroides destabilizes its quaternary structure.

Delipidation of detergent-solubilized cytochrome c oxidase isolated from Rhodobacter sphaeroides (Rbs-CcO) has no apparent structural and/or functional effect on the protein, however affects its resistance against thermal or chemical denaturation. Phospholipase A2 (PLA2) hydrolysis of phospholipids that are co-purified with the enzyme removes all but two tightly bound phosphatidylethanolamines. Replacement of the removed phospholipids with nonionic detergent decreases both thermal stability of the enzyme and its resilience against the effect of chemical denaturants such as urea. In contrast to nondelipidated Rbs-CcO, the enzymatic activity of PLA2-treated Rbs-CcO is substantially diminished after exposure to high (>4 M) urea concentration at room temperature without an alteration of its secondary structure. Absorbance spectroscopy and sedimentation velocity experiments revealed a strong correlation between intact tertiary structure of heme regions and quaternary structure, respectively, and the enzymatic activity of the protein. We concluded that phospholipid environment of Rbs-CcO has the protective role for stability of its tertiary and quaternary structures.

Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles.

Bilayered long- and short-chain phospholipid assemblies, known as bicelles, have been widely used as model membranes in biological studies. However, to date, there has been no demonstration of structural or functional viability for the fundamental mitochondrial electron transport complexes reconstituted into or interacting with bicelles. In the present work, bicelles were formed from the mixture of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, (DHPC)). Isolated from bovine heart, cytochrome c oxidase was successfully incorporated into bicelles. Bicelles and cytochrome c oxidase incorporated into bicelles ("proteobicelles") were characterized by absorption spectroscopy, dynamic light scattering, atomic force microscopy, sedimentation velocity and differential scanning calorimetry. It was demonstrated that at total concentration of phospholipids CL = 24 mM and the molar ratio (q) of long-chain DMPC over short-chain DHPC equal to 0.4, the diameter of bicelles formed at neutral pH is in the range of 30-60 nm with the thickness of bicelles of about 4 nm. Adding cytochrome c oxidase to bicelles unified the size of the resulting proteobicelles to about 160 nm. Cytochrome c oxidase in bicelles was fully reducible by artificial donors of electrons, exhibited "normal" reaction with external ligands, and was fully active. Both, sedimentation velocity analysis and temperature-induced denaturation indicated that enzyme in bicelles is monomeric. We concluded that cytochrome c oxidase in bicelles maintains its structural and functional integrity, and that bicelles can be used for more comprehensive investigation of cytochrome c oxidase and most likely other mitochondrial electron transfer complexes.

Lysozyme stability and amyloid fibrillization dependence on Hofmeister anions in acidic pH.

We have explored an effect of Hofmeister anions, Na2SO4, NaCl, NaBr, NaNO3, NaSCN and NaClO4, on stability and amyloid fibrillization of hen egg white lysozyme at pH 2.7. The stability of the protein was analyzed by differential scanning calorimetry. The Hofmeister effect of the anions was assessed by the parameter dT trs/d[anion] (T trs, transition temperature). We show that dT trs/d[anion] correlates with anion surface tension effects and anion partition coefficients indicating direct interactions between anions and lysozyme. The kinetic of amyloid fibrillization of lysozyme was followed by Thioflavin T (ThT) fluorescence. Negative correlation between dT trs/d[anion] and the nucleation rate of fibrillization in the presence of monovalent anions indicates specific effect of anions on fibrillization rate of lysozyme. The efficiency of monovalent anions to accelerate fibrillization correlates with inverse Hofmeister series. The far-UV circular dichroism spectroscopy and atomic force microscopy findings show that conformational properties of fibrils depend on fibrillization rate. In the presence of sodium chloride, lysozyme forms typical fibrils with elongated structure and with the secondary structure of the ß-sheet. On the other hand, in the presence of both chaotropic perchlorate and kosmotropic sulfate anions, the fibrils form clusters with secondary structure of ß-turn. Moreover, the acceleration of fibril formation is accompanied by decreased amount of the formed fibrils as indicated by ThT fluorescence. Taken together, our study shows Hofmeister effect of monovalent anions on: (1) lysozyme stability; (2) ability to accelerate nucleation phase of lysozyme fibrillization; (3) amount, and (4) conformational properties of the formed fibrils.

Non-two-state thermal denaturation of ferricytochrome c at neutral and slightly acidic pH values.

Thermal denaturation of ferricytochrome c (cyt c) has been methodically studied by absorbance, fluorescence, circular dichroism spectroscopy, viscosimetry and differential scanning calorimetry in pH range from pH 3.5 to 7.5. Thermal transitions have been monitored by intrinsic local probes of heme region such as absorbance at Soret, 620nm and 695nm bands and circular dichroism signals at 417nm. Global conformational changes were analyzed by circular dichroism signal at 222nm, fluorescence of the single tryptophan, reduced viscosity and differential scanning calorimetry. We show that cyt c thermal denaturation above pH ~5 can be described by an apparent two-step transition in which the heme iron stays in a low-spin state. The thermal denaturations of cyt c below pH ~5 proceed in one step to an unfolded highly compact form with a high-spin state of the heme iron. Cyt c conformational plasticity is discussed in regard to its physiological functions.

The kinetic stability of cytochrome C oxidase: effect of bound phospholipid and dimerization.

Thermally induced transitions of the 13-subunit integral membrane protein bovine cytochrome c oxidase (CcO) have been studied by differential scanning calorimetry (DSC) and circular dichroism (CD). Thermal denaturation of dodecyl maltoside solubilized CcO proceeds in two consecutive, irreversible, kinetically driven steps with the apparent transition temperatures at âˆ¼ 51°C and ∼ 61°C (5µM CcO at scan rate of 1.5 K/min). The thermal denaturation data were analyzed according to the Lyubarev and Kurganov model of two consecutive irreversible steps. However, because of the limitation of the model to describe the complex mechanism of the thermal denaturation of CcO, the obtained results were utilized only for comparison purposes of kinetic stabilities of CcO under specific protein concentration (5µM) and scan rate (1.5 K/min). This enabled us to show that both the amphiphilic environment and the self-association state of CcO affect its kinetic stability. Kinetic stabilities of both steps are significantly decreased when all of the phospholipids are removed from CcO by phospholipase A2 (the half-life decreases at 37°C). Conversely, dimerization of CcO induced by sodium cholate significantly increases its kinetic stability of only the first step (the half-life increases at 37°C). Protein concentration-dependent nonspecific oligomerization also indicate mild stabilization of CcO. Both, reversed-phase high-performance liquid chromatography (HPLC) and SDS-PAGE subunit analysis reveal that the first step of thermal denaturation involves dissociation of subunits III, VIa, VIb, and VIIa, whereas the second step is less well defined and most likely involves global unfold and aggregation of the remaining subunits. Electron transport activity of CcO decreases in a sigmoidal manner during the first transition and this dependence is very well described by kinetic parameters for the first step of the thermal transition. Therefore, dissociation of subunit III and/or VIIa is responsible for temperature-induced inactivation of CcO because VIa and VIb can be removed from CcO without affecting the enzyme activity. These results demonstrate an important role of tightly bound phospholipids and oligomeric state (particularly the dimeric form) of CcO for kinetic stability of the protein.

Urea-induced modification of cytochrome c flexibility as probed by cyanide binding.

Cyanide binding to cytochrome c was monitored by absorption spectroscopy from neutral to acidic pH in the presence of urea. These results were compared with acid-induced unfolding at corresponding urea concentration monitored by absorption spectroscopy and circular dichroism. The association rate constant ka increased 20-fold when the concentration of urea was raised from 0M to 6M at neutral pH. However, the secondary structure of the protein was not affected, i.e. there was no striking conformational change in these urea concentrations at neutral pH. At the pH that was very close to the pK of acid-induced unfolding, the ka value reached its maximum (ka,max) in all urea concentrations. Interestingly, the ka,max value increased exponentially with increasing urea concentrations. These results are interpreted in terms of a change in the flexibility of the least stable part of the cyt c structure that is responsible for the Fe-S(Met80) bond disruption and for ligand binding to heme iron.

Elucidating the mechanism of ferrocytochrome c heme disruption by peroxidized cardiolipin.

The interaction of peroxidized cardiolipin with ferrocytochrome c induces two kinetically and chemically distinct processes. The first is a rapid oxidation of ferrocytochrome c, followed by a slower, irreversible disruption of heme c. The oxidation of ferrocytochrome c by peroxidized cardiolipin is explained by a Fenton-type reaction. Heme scission is a consequence of the radical-mediated reactions initiated by the interaction of ferric heme iron with peroxidized cardiolipin. Simultaneously with the heme c disruption, generation of hydroxyl radical is detected by EPR spectroscopy using the spin trapping technique. The resulting apocytochrome c sediments as a heterogeneous mixture of high aggregates, as judged by sedimentation analysis. Both the oxidative process and the destructive process were suppressed by nonionic detergents and/or high ionic strength. The mechanism for generating radicals and heme rupture is presented.

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1.

Cytochrome bc(1) isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc(1) is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc(1) was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc(1) retained nearly full activity, absorption spectra, subunit, and phospholipid composition.

Kinetics of cyanide binding as a probe of local stability/flexibility of cytochrome c.

Effect of anions of the Hofmeister series (thiocyanate, perchlorate, iodide, bromide, nitrate, chloride, sulfate, and phosphate) on local and global stability and flexibility of horse heart ferricytochrome c (cyt c) has been studied. Global stability of cyt c was determined by iso/thermal denaturations monitored by change in ellipticity in the far-UV region and its local stability was determined from absorbance changes in the Soret region. Particularly, relative stability/flexibility of the Met80-heme iron bond has been assessed by analysis of binding of cyanide into the heme iron. Both global and local stabilities of cyt c exhibited monotonous increase induced by a change of anion from chaotropic to kosmotropic species. However, this monotonous dependence was not observed for the rate constants of cyanide association with cyt c. As expected more chaotropic ions induced lower stability of protein and faster binding of cyanide but this correlation was reversed for kosmotropic anions. We propose that the unusual bell-shaped dependence of the rate constant of cyanide association is a result of modulation of Met80-heme iron bond strength and/or flexibility of heme region by Hofmeister anions independently on global stability of cyt c. Further, our results demonstrate sensitivity of cyanide binding to local change in stability/flexibility in the heme region of cyt c.

Correlation of acid-induced conformational transition of ferricytochrome c with cyanide binding kinetics.

A relation between pH-induced conformational transitions of horse heart ferricytochrome c and the kinetics of external ligand coordination to heme iron was investigated by optical spectroscopy, circular dichroism and viscometry. The dependencies of both the association, k (a), and dissociation rate constants of cyanide binding on pH were determined from kinetic measurements. The association rate constant exhibits a bell-shaped form of dependence on pH in the region where this protein unfolds. The maximum of the dependence of k (a) on pH is found to be coincident with the pK values of conformational transitions of ferricytochrome c in solutions with both low and high ionic strengths. This observation is explained in terms of ferricytochrome c unfolding, which is characterized by two processes: the gradual opening of the heme crevice accompanied by the detachment of the axial Met80 and its replacement with a water molecule. The former process enhances the rate, whereas the latter results in the inhibition of the rate of cyanide binding.

Conformational stability and dynamics of cytochrome c affect its alkaline isomerization.

The alkaline isomerization of horse heart ferricytochrome c (cyt c) has been studied by electronic absorption spectroscopy in the presence of the Hofmeister series of anions: chloride, bromide, rhodanide and perchlorate. The anions significantly affect the apparent pK (a) value of the transition in a concentration-dependent manner according to their position in the Hofmeister series. The Soret region of the absorption spectra is not affected by the presence of the salts and shows no significant structural perturbation of the heme crevice. In the presence of perchlorate and rhodanide anions, the cyanide exchange rate between the bulk solvent and the binding site is increased. These results imply higher flexibility of the protein structure in the presence of chaotropic salts. The thermal and isothermal denaturations monitored by differential scanning calorimetry and circular dichroism, respectively, showed a decrease in the conformational stability of cyt c in the presence of the chaotropic salts. A positive correlation between the stability, DeltaG, of cyt c and the apparent pK (a) values that characterize the alkaline transition indicates the presence of a thermodynamic linkage between these conformational transitions. In addition, the rate constant of the cyanide binding and the partial molar entropies of anions negatively correlate with the pK (a) values. This indicates the important role of anion-induced solvent reorganization on the structural flexibility of cyt c in the alkaline transitions.

Determination of the pK for the acid-induced denaturation of ferrocytochrome c.

Optical absorption spectroscopy was used to characterize the acid-induced conformational transition of horse heart ferrocytochrome c in the presence of urea. By using linear extrapolation to zero denaturant concentration, an apparent pK value for denaturation was found to be 0.86 +/- 0.07 at 25 degrees C. Visible absorption spectra in the presence of high urea concentration indicate that the dominant population is a high-spin, five-coordinate form under acidic conditions. Ferricytochrome c, used as a model reference system, shows a linear dependence of pK values versus urea concentration in the range from 0 to 4.1 M. Our data also indicate that even at a pH below 2 the iron-sulfur bond in ferrocytochrome c is present.