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.     

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.     

Comparison of changes in succinate dehydrogenase activity in blood lymphocytes and modification of radiosensitivity by exogenous hypoxia

Radioprotective efficiency of gas hypoxic mixtures (GHM) containing 5-12% of oxygen and the rate of the reaction of succinate dehydrogenase (VSDG) activity in peripheral blood lymphocytes upon breathing GHM were comparatively studied in rats and dogs. VSDG was 4393.5 (%O2)-2.58 and 130.76 (%O2)-1.42 in dogs and rats respectively. Taking into account that DMF in rats is a function of oxygen concentration in the mixture one can obtain a formula for determining a dose modifying factor (DMF) as a function of the rate of SDG activity reaction.     

Radiosensitivity during the irradiation of animals in an altered gaseous environment. A comparative study on mice and rats of the radiation-protective effect of oxygen-deficient gas mixtures

The radioprotective effect of gas hypoxic mixtures containing 5, 7, 8, 10 and 15% of oxygen on mice and rats was comparatively studied. The dependence of DMF upon oxygen concentration in the mixture was approximated by a hyperbolic function similar to the dependence of the radiomodifying effect of circulatory hypoxia caused by radioprotective agents of the indolylalkylamine series.     

Continuous stereospecific Δ-3-keto-steroid reduction by PAAH bead-entrapped Clostridium paraputrificum cells

The development of a continuous anaerobic process for stereospecific Δ⁴-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ⁴, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle—in the presence of the cosolvent—resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested.     

Fixation and stabilization of Escherichia coli cells displaying genetically engineered cell surface proteins

A large biotechnological potential is inherent in the display of proteins (e.g., enzymes, single-chain antibodies, on the surface of bacterial cells) (Georgiou et al., 1993). Applications such as immobilized whole-cell biocatalysts or cellular adsorbents require cell fixation to prevent disintegration, stabilization of the anchored protein from leakage, denaturation or proteolysis, and total loss of cell viability, preventing medium and potential product contamination with cells. In this article we describe the adaptation of a simple two-stage chemical crosslinking procedure based on "bi-layer encagement" (Tor et al., 1989) for stabilizing Escherichia coli cells expressing an Lpp-OmpA (46-159)-beta-lactamase fusion that displays beta-lactamase on the cell surface. Bilayer crosslinking and coating the bacteria with a polymeric matrix is accomplished by treating the cells first with either glutaraldehyde or polyglutaraldehyde, followed by secondary crosslinking with polyacrylamide hydrazide. These treatments resulted in a 5- to 25-fold reduction of the thermal inactivation rate constant at 55 degrees C of surface anchored beta-lactamase and completely prevented the deterioration of the cells for at least a week of storage at 4 degrees C. The stabilization procedure developed paves the way to scalable biotechnological applications of E. coli displaying surface anchored proteins as whole-cell biocatalysts and adsorbents. (c) 1996 John Wiley & Sons, Inc.     

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.     

Action of surfactants on egg lecithin liposomes

The lytic action of several homologous series of surfactants including N-acyl derivatives of the Na-salt of amino acids on the egg lecithin multilamellar liposomes was examined. The affinity for the lipid membrane and the solubilising capacity of the agents were estimated. The contribution of a CH₂ group and that of the polar head group of surfactants to the free energy of the agent's binding to the membrane were evaluated. The results obtained indicate that the contribution of a CH₂ group to the free binding energy depends on the nature of the surfactants' head group. This dependence is attributed to either various localisation of the agent's molecules in the lipid bilayer or to different properties of the agent's hydrocarbon tails. The contributions of the head groups of the surfactants are assumed to reflect the affinity of these head groups for the lecithin polar head group at the membrane interface. The results obtained indicate some degree of specificity involved in the interactions of the head groups.