Influence of cation-π interactions to the structural stability of phycocyanin proteins: A computational study.

The influences of cation-π interactions in phycocyanin proteins and their environmental preferences were analyzed. The number of interactions formed by arginine showed to be higher than those formed by the lysine in the cationic group, while histidine is comparatively higher than phenylalanine and N-terminal residue in the π group. Arg-Tyr and Arg-Phe interacting pairs are predominant among the various pairs analyzed. Cation-π interactions are distance-dependent and can be realized above a wider area above the π ring. We analyzed the energy contribution resulting from cation-π interactions using ab initio calculations. The energy contribution resulting from the most frequent cation-π interactions was in the lower range of strong hydrogen bonds. The results showed that, while most of their interaction energies lay ranged from - 2 to - 8 kcal/mol, those energies could be up to -12- 12 kcal/mol. Stabilization centers for these proteins showed that all residues found in cation-π interactions are important in locating one or more of such centers. In the cation-π interacting residues, 54% of the amino acid residues involved in these interactions might be conserved in phycocyanins. From this study, we infer that cation-π forming residues play an important role in the stability of the multiply commercially used phycocyanin proteins and could help structural biologists and medicinal chemists to design better and safer drugs.

Antipsychotic clozapine binding to alpha-2-macroglobulin protects interacting partners against oxidation and preserves the anti-proteinase activity of the protein.

In this study, the interaction between clozapine, an atypical antipsychotic drug, and alpha-2-macroglobulin (α2M), a multipurpose anti-proteinase, was investigated under simulated (patho) physiological conditions using multiple spectroscopic techniques and molecular modeling. It was found that α2M binds clozapine with a moderate affinity (the binding constant of 0.9 × 105 M-1 at 37 °C). The preferable binding site for both clozapine's atropisomers was revealed to be a large pocket at the interface of C and D monomer subunits of the protein. Hydrogen bonds and the hydrophobic effect were proposed as dominant forces in complex formation. The binding of clozapine did not induce significant conformational change of the protein, as confirmed by virtually unaltered α2M secondary structure and anti-proteinase activity. However, both clozapine and α2M shielded each other from the deleterious influence of strong oxidants: sodium hypochlorite and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). Moreover, clozapine in a concentration range that is usually targeted in the plasma during patients' treatment effectively protected the anti-proteinase activity of α2M under AAPH-induced free radical overproduction. Our results suggest that the cooperation between α2M and clozapine may be a path by which these two molecules synergistically protect neural tissue against injury caused by disturbed proteostasis or oxidative stress.

Nutraceutical phycocyanobilin binding to catalase protects the pigment from oxidation without affecting catalytic activity.

Phycocyanobilin is a dark blue linear tetrapyrrole chromophore covalently attached to protein subunits of phycobiliproteins present in the light-harvesting complexes of the cyanobacteria Arthrospira platensis (Spirulina "superfood"). It shows exceptional health-promoting properties and emerging use in various fields of bioscience and industry. This study aims to examine the mutual impact of phycocyanobilin interactions with catalase, a life-essential antioxidant enzyme. Fluorescence quenching experiments demonstrated moderate binding (Ka of 3.9 × 104 M-1 at 25 °C; n = 0.89) (static type), while van't Hoff plot points to an enthalpically driven ligand binding (ΔG = -28.2 kJ mol-1; ΔH = -41.9 kJ mol-1). No significant changes in protein secondary structures (α-helix content ~22%) and thermal protein stability in terms of enzyme tetramer subunits (Tm ~ 64 °C) were detected upon ligand binding. Alterations in the tertiary catalase structure were found without adverse effects on enzyme activity (~2 × 106 IU/mL). The docking study results indicated that the ligand most likely binds to amino acid residues (Asn141, Arg 362, Tyr369 and Asn384) near the cavity between the enzyme homotetramer subunits not related to the active site. Finally, complex formation protects the pigment from free-radical induced oxidation (bleaching), suggesting possible prolongation of its half-life and bioactivity in vivo if bound to catalase.

Computational Analysis of Non-covalent Interactions in Phycocyanin Subunit Interfaces.

Protein-protein interactions are an important phenomenon in biological processes and functions. We used the manually curated non-redundant dataset of 118 phycocyanin interfaces to gain additional insight into this phenomenon using a robust inter-atomic non-covalent interaction analyzing tool PPCheck. Our observations indicate that there is a relatively high composition of hydrophobic residues at the interfaces. Most of the interface residues are clustered at the middle of the range which we call "standard-size" interfaces. Furthermore, the multiple interaction patterns founded in the present study indicate that more than half of the residues involved in these interactions participate in multiple and water-bridged hydrogen bonds. Thus, hydrogen bonds contribute maximally towards the stability of protein-protein complexes. The analysis shows that hydrogen bond energies contribute to about 88 % to the total energy and it also increases with interface size. Van der Waals (vdW) energy contributes to 9.3 %±1.7 % on average in these complexes. Moreover, there is about 1.9 %±1.5 % contribution by electrostatic energy. Nevertheless, the role by vdW and electrostatic energy could not be ignored in interface binding. Results show that the total binding energy is more for large phycocyanin interfaces. The normalized energy per residue was less than -16 kJ mol-1 , while most of them have energy in the range from -6 to -14 kJ mol-1 . The non-covalent interacting residues in these proteins were found to be highly conserved. Obtained results might contribute to the understanding of structural stability of this class of evolutionary essential proteins with increased practical application and future designs of novel protein-bioactive compound interactions.

Opposite clozapine and ziprasidone effects on the reactivity of plasma albumin SH-group are the consequence of their different binding properties dependent on protein fatty acids content.

Antipsychotic drugs interfere with the antioxidant defense system provoking complex and often toxicological effects. Here we examined differences in plasma albumin reduced free thiol (SH) group content and its reactivity as a consequence of clozapine (CLZ) and ziprasidone (ZIP) binding. Chronic administration of CLZ reduced, whereas treatment with ZIP increased albumin-SH content in rats. Regardless of the ratio of stearic acid (SA) bound to protein, in vitro binding of ZIP to human serum albumin (HSA) increased both the SH group level and reactivity. In contrast, the effect of CLZ on HSA-SH reactivity was dependent on HSA to SA molar ratio. CLZ binding was accompanied by an increase in HSA-SH reactivity in samples with normal, but a reduction of its reactivity level with higher SA/HSA ratio, compared to drug-free samples. We demonstrate by steady-state fluorescence quenching studies that an increase in SA binding to HSA is associated with a significant reduction of binding constant for both antipsychotics. In addition, this is the first report of quantitative characterization of ZIP binding to HSA. Our findings suggest that albumin-SH content and reactivity is modulated by ZIP towards an increased antioxidant defense capacity in circulation, as opposed to CLZ, which can contribute to the safer, more effective treatment of schizophrenia.

Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin, a blue-colored biliprotein of microalga Spirulina.

C-phycocyanin, the major protein of cyanobacteria Spirulina, possesses significant antioxidant, anti-cancer, anti-inflammatory and immunomodulatory effects, ascribed to covalently attached linear tetrapyrrole chromophore phycocyanobilin. There are no literature data about structure and biological activities of released peptides with bound chromophore in C-phycocyanin digest. This study aims to identify chromopeptides obtained after pepsin digestion of C-phycocyanin and to examine their bioactivities. C-phycocyanin is rapidly digested by pepsin in simulated gastric fluid. The structure of released chromopeptides was analyzed by high resolution tandem mass spectrometry and peptides varying in size from 2 to 13 amino acid residues were identified in both subunits of C-phycocyanin. Following separation by HPLC, chromopeptides were analyzed for potential bioactivities. It was shown that all five chromopeptide fractions have significant antioxidant and metal-chelating activities and show cytotoxic effect on human cervical adenocarcinoma and epithelial colonic cancer cell lines. In addition, chromopeptides protect human erythrocytes from free radical-induced hemolysis in antioxidative capacity-dependant manner. There was a positive correlation between antioxidative potency and other biological activities of chromopeptides. Digestion by pepsin releases biologically active chromopeptides from C-phycocyanin whose activity is mostly related to the antioxidative potency provided by chromophore.

Contribution of cation-π interactions to the stability of Sm/LSm oligomeric assemblies.

In this work, we have analyzed the influence of cation-π interactions to the stability of Sm/LSm assemblies and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while histidine is comparatively higher than phenylalanine and tyrosine in the π group. Arg-Tyr interactions are predominant among the various pairs analyzed. The furcation level of multiple cation-π interactions is much higher than that of single cation-π interactions in Sm/LSm interfaces. We have found hot spot residues forming cation-π interactions, and hot spot composition is similar for all aromatic residues. The Arg-Phe pair has the strongest interaction energy of -8.81 kcal mol(-1) among all the possible pairs of amino acids. The extent of burial of the residue side-chain correlates with the ΔΔG of binding for residues in the core and also for hot spot residues cation-π bonded across the interface. Secondary structure of the cation-π residues shows that Arg and Lys preferred to be in strand. Among the π residues, His prefers to be in helix, Phe prefers to be in turn, and Tyr prefers to be in strand. Stabilization centers for these proteins showed that all the five residues found in cation-π interactions are important in locating one or more of such centers. More than 50 % of the cation-π interacting residues are highly conserved. It is likely that the cation-π interactions contribute significantly to the overall stability of Sm/LSm proteins.

Contribution of anion-π interactions to the stability of Sm/LSm proteins.

We have analyzed the influence of anion-π interactions to the stability of Sm/LSm assemblies. The side chain of Glu is more likely to be in anion-π interactions than Asp. Phe has the highest occurrence in these interactions than the other two π residues. Among the anion-π residue pairs, Glu-Phe residue pair showed the maximum number of anion-π. We have found hot-spot residues forming anion-π interactions, and Glu-Phe is the most common hot-spot interacting pair. The significant numbers of anion-π interacting residues identified in the dataset were involved in the formation of multiple anion-π interactions. More than half of the residues involved in these interactions are evolutionarily conserved. The anion-π interaction energies are distance and orientation dependent. It was found that anion-π interactions showed energy less than -15 kcal mol(-1), and most of them have energy in the range -2 to -9 kcal mol(-1). Solvent accessibility pattern of Sm/LSm proteins reveals that all of the interacting residues are preferred to be in buried regions. Most of the interacting residues preferred to be in strand. A significant percentage of anion-π interacting residues are located as stabilization centers and thus might provide additional stability to these proteins. The simultaneous interaction of anions and cations on different faces of the same π-system has been observed. On the whole, the results presented in this work will be very useful for understanding the contribution of anion-π interaction to the stability of Sm/LSm proteins.