Srđan Đ. Stojanović

Research Article: Halogen bonding in complexes of proteins and non-natural amino acids

In this work, we have analyzed the influence of halogen bonding to the stability of 44 complexes of proteins and non-natural amino acids. Fluorine- and chlorine-containing non-natural amino acids are more prevalent in the dataset, and an even larger number of contacts made by iodine-containing ligands are found. Only few halogen bonds with the hydroxyl oxygens and carboxylate side chains are found in the dataset. Halogen bonds with the nitrogen-containing side chains have higher occurrence than other acceptors. Backbone carbonyl oxygens and nitrogens are to a substantial extent involved in our dataset. We have observed a small percentage of interactions involving water as hydrogen bond donors. Additionally, most of the interacting residues comprising the interfaces also show a great degree of conservation. There is a clear interaction hot spot at distances of 3.5-3.7 Å and Θ1 angles of 100-120°. There is also a cluster of contacts featuring short distances (2.6-2.9 Å) but only nearly optimal Θ1 angles (140-160°). 51.3% of stabilizing residues are involved in building halogen bonds with the non-natural amino acids. We discovered three types of structural motifs significantly over-represented: beta-turn-ir, beta-turn-il and niche-4r. The halogen-bonding statistics of the dataset do not show any preference for α-helices (36%), β-sheets (36%), or turns/coils (28%) structures. Most of the amino acid residues that were involved in halogen bonds prefer to be in the solvent excluded environment (buried). Furthermore, we have shown that in amino acid-protein complexes halogen atoms can sometimes be involved in hydrogen bonding interactions with hydrogen bonding-donors. The results from this study might be used for the rational design of halogenated ligands as inhibitors and drugs, and in biomolecular engineering.

Effects of infection intensity with Strongyloides papillosus and albendazole treatment on development of oxidative/nitrosative stress in sheep.

The objective of this study was to estimate and evaluate oxidative/nitrosative stress parameters in sheep infected with Strongyloides papillosus and after antihelminthic treatment with albendazole (ABZ). This parasite, especially during development stages can seriously damage parenchaematous organs during migration within the host. The presence of parasites leads to increased productions of reactive oxygen species (ROS) and reactive nitrogen species (RNS). It is also well known that certain drugs can be very harmful for the delicate oxidant-antioxidant equilibrium, provoking oxidative stress during their biotransformation. ABZ is a broad spectrum antihelminthic drug, frequently used in veterinary medicine for therapy of parasitic infections. The current research was performed on female Württemberg sheep (n=48). The distribution of parasites in sheep was evaluated using the native smear coprological technique, by sedimentation and flotation methods, revealing the presence of S. papillosus. The degree of infection intensity per sheep was quantitatively established by the method of McMaster, the animals having been divided into three groups according to the intensity of infection; mild, moderate and high. The control group consisted of sheep negative to the parasites. After determining the type of parasite infection, the sheep were treated with ABZ, per orally, in single doses of 5mg/kg per body weight. Sampling of feces for parasitological and blood for biochemical assaying was performed on the 0 and 21st day after treatment with ABZ. The oxidative stress parameters were measured for catalase activity (CAT), the red cell membrane damage by level of malondialdehyde (MDA), while carbonyl and thiol plasma protein group concentrations were used as indicators of the degree of protein oxidative modification. The activity of Cu,Zn-superoxide dismutase (SOD) and relative distribution of lactate dehydrogenase (LDH(1)-LDH(5)) activity were determined electrophoretically. The distribution of LDH isoenzymes in sheep moderately and highly infected with S. papillosus revealed that the parasite induced damage to the myocardial (LDH(2)), lung (LDH(3)) and liver cells (LDH(5)) in infected animals, while ABZ treatment only damaged liver cells (LDH(5)). The MDA concentration revealed that lipid peroxidation increased both in the presence of parasites and the antihelminthic formulation tested (p<0.001) when compared to the control sheep, while the increase of carbonyl concentration (p<0.001), as well as the observed decrease of thiol concentration (p<0.001) indicated significant oxidative damage of plasma proteins in experimental sheep, when compared to the control animals. Our results indicate that S. papillosus induces oxidative/nitrosative stress in sheep. The antihelminthic treatment with ABZ further promotes the disbalance of oxidative-antioxidative equilibrium in all tested sheep.

π–π and cation–π interactions in protein–porphyrin complex crystal structures

In this study we have described the π–π and cation–π interactions between the porphyrin ring and the protein part of porphyrin-containing proteins to better understand their stabilizing role. The number of π–π interactions was higher than that of cation–π interactions in the same set of proteins studied. The pyrrole groups of one porphyrin can be involved in π–π interactions with π systems of another porphyrin in the protein. We have found 5.1% cation–π interactions between porphyrin Fe2+ metal cations and π systems of surrounding amino acids as well as the pyrrole rings of other porphyrins. We observed that most of the π–π interactions have an energy in the range −0.5 to −2.0 kcal mol−1, while the cation–π interactions showed an energy in the range −2 to −4 kcal mol−1. Further, an appreciable number of metal/cation–π interaction pairs have an energy in the range −6 to −13 kcal mol−1. The preferred parallel-stacked orientation is found to be more stable than a T-shaped structure for the full set of π–π interaction pairs. In the case of cation–π interactions, it was found that 44% of the cation–π interactions involved planar stacking, 37% of the interactions belonged to the oblique category, and the remaining 19% of the interactions were of the orthogonal type. The separation distance between the cation group and the aromatic ring decreases as the interplanar angle decreases. Furthermore, in the present study we have found that 10.4% of π residues and 3.9% of cationic residues were found to have one or more stabilization centers. Amino acids deployed in the environment of porphyrin rings are deposited in helices and coils. The results from this study might be used for structure-based porphyrin protein prediction and as scaffolds for future porphyrin-containing protein design.

Research article: Cation-π interactions in high resolution protein-RNA complex crystal structures

In this work, we have analyzed the influence of cation-π interactions to the stability of 59 high resolution protein-RNA complex crystal structures. The total number of Lys and Arg are similar in the dataset as well as the number of their interactions. On the other hand, the aromatic chains of purines are exhibiting more cation-π interactions than pyrimidines. 35% of the total interactions in the dataset are involved in the formation of multiple cation-π interactions. The multiple cation-π interactions have been conserved more than the single interactions. The analysis of the geometry of the cation-π interactions has revealed that the average distance (d) value falls into distinct ranges corresponding to the multiple (4.28 Å) and single (5.50 Å) cation-π interactions. The G-Arg pair has the strongest interaction energy of -3.68 kcal mol(-1) among all the possible pairs of amino acids and bases. Further, we found that the cation-π interactions due to five-membered rings of A and G are stronger than that with the atoms in six-membered rings. 8.7% stabilizing residues are involved in building cation-π interactions with the nucleic bases. There are three types of structural motifs significantly over-represented in protein-RNA interfaces: beta-turn-ir, niche-4r and st-staple. Tetraloops and kink-turns are the most abundant RNA motifs in protein-RNA interfaces. Amino acids deployed in the protein-RNA interfaces are deposited in helices, sheets and coils. Arg and Lys, involved in cation-π interactions, prefer to be in the solvent exposed surface. The results from this study might be used for structure-based prediction and as scaffolds for future protein-RNA complex design.

Anion–π interactions in protein–porphyrin complexes

In this work, we have analyzed the influence of anion–π interactions on the stability of high resolution protein–porphyrin complex crystal structures. The anion–π interactions are distance and orientation dependent. Results of ab initio calculations of stabilization energies showed that they lie mostly in the range from −2 to −4 kcal mol−1 with some of the anion–π interactions having stabilization energies of up to −16 kcal mol−1. In the anionic group, the numbers of anion–π interactions involving Asp and Glu are similar, while His is more often involved in these interactions than other aromatic residues. Furthermore, our study showed that in the dataset used about 70% of the investigated anion–π interactions are in fact multiple anion–π interactions. Our results suggest that interacting residues are predominantly located in buried and partially buried regions. The secondary structure of the anion–π interaction involving residues shows that most of the interacting residues preferred to be in helix conformations. Significant numbers of aromatic residues involved in anion–π interactions have one or more stabilization centers, providing additional stability to the protein–porphyrin complexes. The conservation patterns indicate that more than half of the residues involved in these interactions are evolutionarily conserved, indicating that the contribution of the anion–π interaction is an important factor for the structural stability of the investigated protein–porphyrin complexes.

A reexamination of correlations of amino acids with particular secondary structures.

Using the data from Protein Data Bank the correlations of primary and secondary structures of proteins were analyzed. The correlation values of the amino acids and the eight secondary structure types were calculated, where the position of the amino acid and the position in sequence with the particular secondary structure differ at most 25. The diagrams describing these results indicate that correlations are significant at distances between −9 and 10. The results show that the substituents on Cβ or Cγ atoms of amino acid play major role in their preference for particular secondary structure at the same position in the sequence, while the polarity of amino acid has significant influence on α-helices and strands at some distance in the sequence. The diagrams corresponding to polar amino acids are noticeably asymmetric. The diagrams point out the exchangeability of residues in the proteins; the amino acids with similar diagrams have similar local folding requirements.

Protein subunit interfaces: a statistical analysis of hot spots in Sm proteins

The distinguishing property of Sm protein associations is very high stability. In order to understand this property, we analyzed the interfaces and compared the properties of Sm protein interfaces with those of a test set, the Binding Interface Database (BID). The comparison revealed that the main differences between the interfaces of Sm proteins and those of the BID set are the content of charged residues, the coordination numbers of the residues, knowledge-based pair potentials, and the conservation scores of hot spots. In Sm proteins, the interfaces have more hydrophobic and fewer charged residues than the surfaces, which is also the case for the BID test set and other proteins. However, in the interfaces, the content of charged residues in Sm proteins (26%) is substantially larger than that in the BID set (22%). Hot spots are residues that make up a small fraction of the interfaces, but they contribute most of the binding energy. These residues are critical to protein–protein interactions. Analyses of knowledge-based pair potentials of hot spot and non-hot spot residues in Sm proteins show that they are significantly different; their mean values are 31.5 and 11.3, respectively. In the BID set, this difference is smaller; in this case, the mean values for hot spot and non-hot spot residues are 20.7 and 12.4, respectively. Hence, the pair potentials of hot spots differ significantly for the Sm and BID data sets. In the interfaces of Sm proteins, the amino acids are tightly packed, and the coordination numbers are larger in Sm proteins than in the BID set for both hot spots and non-hot spots. At the same time, the coordination numbers are higher for hot spots; the average coordination number of the hot spot residues in Sm proteins is 7.7, while it is 6.1 for the non-hot spot residues. The difference in the calculated average conservation score for hot spots and non-hot spots in Sm proteins is significantly larger than it is in the BID set. In Sm proteins, the average conservation score for the hot spots is 7.4. Hot spots are surrounded by residues that are moderately conserved (5.9). The average conservation score for the other interface residues is 5.6. The conservation scores in the BID set do not show a significant distinction between hot and non-hot spots: the mean values for hot and non-hot spot residues are 5.5 and 5.2, respectively. These data show that structurally conserved residues and hot spots are significantly correlated in Sm proteins.

Implications of oxidative stress in occupational exposure to lead on a cellular level

The aim of this study was to determine oxidative alterations leading to cellular dysfunctions in Pb-exposed subjects by evaluating damage to all major classes of biomolecules in the cell, lipid peroxidation, protein and DNA damage and determine relationships between parameters of Pb toxicity and specific biomarkers of oxidative damage. Analysis was conducted of smelter workers with high blood Pb and urine aminolevulinic acid levels and slightly elevated values of coproporphyrin and erythrocyte protoporphyrin IX. Significant decreases of thiol groups and increases in carbonyl groups as protein degradation end products, and of nitrite were detected. Elevated rates of lipid peroxidation and rises in the activities of the antioxidant enzymes Cu–Zn superoxide dismutase and catalase were also observed. Both enzymes showed positive correlations with the blood lead levels and urine coproporphyrin, while thiol groups correlated negatively with the same indices. The genotoxic potential of lead was manifested through an increased number of DNA-damaged cells. Increased activities of serum lactate dehydrogenase isoenzymes indicated cellular damage in the lungs, kidneys, and liver. These lead-induced impairments should be taken into consideration in the assessment of Pb-related health hazards.

Contribution of Non-Canonical Interactions to the Stability of Sm/LSm Oligomeric Assemblies

The distinguishing property of Sm/LSm protein assemblies is their high stability. In order to better understand the nature of Sm/LSm protein oligomers in this study we have analyzed the contribution of non‐canonical interactions to the stability of assemblies. The predominant types of non‐canonical interactions at Sm/LSm protein interfaces are CH⋅⋅⋅O, and CH⋅⋅⋅N interactions represented at interfaces. Our results show low percentages of XH–π and non‐canonical interactions involving sulfur atoms, while the backbone groups were less frequently involved. The data show a high percentage of non‐canonical interactions in interfaces formed by charged residues with Lys and Arg, these being the major charged donors. The main chain non‐canonical interactions might be slightly more linear than the side chain interactions, and they have somewhat shorter median distances. Comparing the stabilizing amino acid residues with amino acids which build non‐canonical interactions at interfaces shows that certain amino acids like Phe, Pro, His and Tyr are involved with a high percentage. The high conservation score of amino acids that are involved in non‐canonical interactions in protein interfaces is an additional strong argument for their importance in the stabilization of Sm/LSm protein assemblies.

CaNa2EDTA chelation attenuates cell damage in workers exposed to lead--a pilot study.

Lead induced oxidative cellular damage and long-term persistence of associated adverse effects increases risk of late-onset diseases. CaNa2EDTA chelation is known to remove contaminating metals and to reduce free radical production. The objective was to investigate the impact of chelation therapy on modulation of lead induced cellular damage, restoration of altered enzyme activities and lipid homeostasis in peripheral blood of workers exposed to lead, by comparing the selected biomarkers obtained prior and after five-day CaNa2EDTA chelation intervention. The group of smelting factory workers diagnosed with lead intoxication and current lead exposure 5.8 ± 1.2 years were administered five-day CaNa2EDTA chelation. Elevated baseline activity of antioxidant enzymes Cu, Zn-SOD and CAT as well as depleted thiols and increased protein degradation products-carbonyl groups and nitrites, pointing to Pb induced oxidative damage, were restored toward normal values following the treatment. Lead showed inhibitor potency on both RBC AChE and BChE in exposed workers, and chelation re-established the activity of BChE, while RBC AChE remained unaffected. Also, genotoxic effect of lead detected in peripheral blood lymphocytes was significantly decreased after therapy, exhibiting 18.9% DNA damage reduction. Administration of chelation reversed the depressed activity of serum PON 1 and significantly decreased lipid peroxidation detected by the post-chelation reduction of MDA levels. Lactate dehydrogenase LDH1-5 isoenzymes levels showed evident but no significant trend of restoring toward normal control values following chelation. CaNa2EDTA chelation ameliorates the alterations linked with Pb mediated oxidative stress, indicating possible benefits in reducing health risks associated with increased oxidative damage in lead exposed populations.