Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Inactivation and oxidative modification of Cu,Zn superoxide dismutase by stimulated neutrophils: the appearance of new catalytically active structures.

Bovine superoxide dismutase (SOD) was inactivated during incubation with phorbol myristate acetate-stimulated neutrophils. In addition, stimulated neutrophils were able to disrupt the SOD structure. Inactivation and structural damage were dependent on the action of hypochlorous acid, an oxidant generated by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils. Incubation of SOD with stimulated neutrophils lead to long-wavelength fluorescence (ex, 350 nm; em, 450 nm) and the appearance of new structural forms with other isoelectric points. These additional forms possess catalytic activity. Generation of catalytically active new forms of SOD demonstrates the inaccessibility of the active centre of SOD to hypochlorite and may be a reason for the successful application of SOD during anti-inflammatory therapy.     

Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

Aggregation of membrane proteins of E. coli cells after treatment with singlet oxygen

It was found that interaction of 1O2 with bacterial membranes of E. coli cells results in covalent binding and aggregation of membrane proteins. This process was shown to be inhibited by water-soluble free radical scavengers, e. g., 1O2 quenchers (cysteine, sodium azide, histidine), of which the latter afforded the strongest inhibition. No protective effect of the fat-soluble free radical scavenger ionol (BHT) on membrane protein aggregation was observed. It was assumed that the main role in oxidative destruction of bacterial membranes (in contrast to membranes from animal sources) is ascribed to processes which are not coupled to lipid peroxidation.     

Comparison of the heme electron state of reduced cytochrome P450 and P420 in equilibrium and non-equilibrium protein conformations. The nature of the protein heme ligand

Magnetic circular dichroism (MCD) spectra of reduced cytochromes P450 and P420 in equilibrium and non-equilibrium protein conformations are compared at 4.2 K for the 350-800 spectral region. Non-equilibrium forms have been produced by photolysis of CO-complexes at 4.2 K. The differences between MCD spectra of proteins in equilibrium and non-equilibrium conformations, in particular for the visible region, show clearly the structural changes in the heme iron coordination sphere to occur on ligand binding. The comparison of the Soret MCD spectra of reduced proteins in their equilibrium and non-equilibrium forms with those of other high-spin ferrous hemoproteins suggest that mercaptide (RS-) is the protein ligand of the heme iron in reduced P450, as well as in its CO-complex, and that imidazole of histidine is the fifth ligand of the iron both in reduced P420 and its CO-complex. The thermal recombination of the photoproducts with CO have been studied. When temperature rises from 4.2 to 77 K for two hours both proteins have similar temperature characteristics during the recombination processes. The recombination begins at T approximately equal to 10 K and is completed at approximately equal to 50 K. The temperature at which half of the total photolyzed molecules are restored to the CO-form is equal to 25 K. For products of photolysis of CO-complexes of myoglobin and hemoglobin under the same heating conditions these temperatures are equal to 35 and 23 K respectively. Thus, the photoproducts of P450, P420 and hemoglobin have similar parameters of low-temperature recombination and the kinetics of this process is faster than for photodissociated myoglobin.     

Carnosine as a potential scavenger of oxidants generated by stimulated neutrophils

Luminol-dependent chemiluminescence of PMA-stimulated human neutrophils decrease more than by 50% in the presence of physiological concentrations of carnosine (20 mM). This inhibition is the result of carnosine ability to scavenge hypochlorite (OCl-), since carnosine exerts a similar effect on chemiluminescence produced by myeloperoxidase-H2O2-Cl- and OCl(-)-H2O2 systems. The previously undocumented property of this dipeptide to scavenge active oxygen species requires further experiments.     

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.     

Enzymatic synthesis of β-lactam antibiotics: A thermodynamic background

The equilibrium constants and the respective standard Gibbs energy changes for hydrolysis of some β-lactam antibiotics have been determined. Native and immobilized penicillin amidase (EC 3.5.1.11) from Escherichia coli has been used as a catalyst. The values of standard Gibbs energy changes corresponding to the pH-independent product of equilibrium concentrations (ΔG⁰_c = ? RT ln K_c) have been calculated. The differences in the structure of the antibiotics nucleus hardly ever affect the value of the pH-independent component of the standard Gibbs energy change (ΔG⁰_c) and value of apparent standard Gibbs energy change at a fixed pH (ΔG^0′_c). At the same time, the value of ΔG⁰_c is more sensitive to the structure of the acyl moiety of the antibiotic; when ampicillin is used instead of benzylpenicillin, ΔG⁰_c increases by ～6.3 kJ mol^?1 (1.5 kcal mol^?1). pH-dependences of the apparent standard Gibbs energy changes for hydrolysis of β-lactam antibiotics have been calculated. The pH-dependences of ΔG^0′_c for hydrolysis of all β-lactam antibiotics have a similar pattern. The thermodynamic pH optimum of the synthesis of these compounds is in the acid pH range (pH < 5.0). The breakage of the β-lactam ring leads to a sharp decrease in the ΔG^0′_c value and a change in the pattern of the pH-dependence. For example, at pH 5.0 ΔG^0′_c decreases from 14.4 kJ mol^?1 for benzylpenicillin to ?1.45 kJ mol^?1 for benzylpenicilloic acid. The reason for these changes is mainly a considerable increase in the pK of the amino group of the nucleus of the antibiotic and, as a consequence, a decrease in the component of standard Gibbs energy change, corresponding to the ionization of the system. The thermodynamic potentials of the enzymatic synthesis of semisynthetic penicillins and cephalosporins on the basis of both free acids and their derivatives (N-acylated amino acids, esters) are discussed. It is shown that with esters of the acids, a high yield of the antibiotic can, in principle, be achieved at higher pH values.