Burial of nonpolar surface area and thermodynamic stabilization of globins as a function of chain elongation

Proteins are biosynthesized from N to C terminus before they depart from the ribosome and reach their bioactive state in the cell. At present, very little is known about the evolution of conformation and the free energy of the nascent protein with chain elongation. These parameters critically affect the extent of folding during ribosome‐assisted biosynthesis. Here, we address the impact of vectorial amino acid addition on the burial of nonpolar surface area and on the free energy of native‐like structure formation in the absence of the ribosomal machinery. We focus on computational predictions on proteins bearing the globin fold, which is known to encompass the 3/3, 2/2, and archaeal subclasses. We find that the burial of nonpolar surface increases progressively with chain elongation, leading to native‐like conformations upon addition of the last C‐terminal residues, corresponding to incorporation of the last two helices. Additionally, the predicted folding entropy for generating native‐like structures becomes less unfavorable at nearly complete chain lengths, suggesting a link between the late burial of nonpolar surface and water release. Finally, the predicted folding free energy takes a progressive favorable dip toward more negative values, as the chain gets longer. These results suggest that thermodynamic stabilization of the native structure of newly synthesized globins during translation in the cell is significantly enhanced as the chain elongates. This is especially true upon departure of the last C‐terminal residues from the ribosomal tunnel, which hosts ca., 30–40 amino acids. Hence, we propose that release from the ribosome is a crucial step in the life of single‐domain proteins in the cell. Proteins 2014; 82:2318–2331. © 2014 Wiley Periodicals, Inc.     

Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

Phenylalanine ammonia-lyase and cell wall peroxidase are cooperatively involved in the extensive formation of ferulate network in cell walls of developing rice shoots

The relationship between the formation of cell wall-bound ferulic acid (FA) and diferulic acid (DFA) and the change in activities of phenylalanine ammonia-lyase (PAL) and cell wall-bound peroxidase (CW-PRX) was studied in rice shoots. The length and the fresh mass of shoots increased during the growth period from day 4 to 6, while coleoptiles ceased elongation growth on day 5. The amounts of FA and DFA isomers as well as cell wall polysaccharides continued to increase during the whole period. The activities of PAL and CW-PRX greatly increased in the same manner during the period. There were close correlations between the PAL activity and ferulate content or between the CW-PRX activity and DFA content. The expression levels of investigated genes for PAL and putative CW-PRX showed good accordance with the activities of these enzymes. These results suggest that increases in PAL and CW-PRX activities are cooperatively involved in the formation of ferulate network in cell walls of rice shoots and that investigated genes may be, at least in part, associated with the enzyme activities. The substantial increase in such network probably causes the maturation of cell walls and thus the cessation of elongation growth of coleoptiles.     

The roles of veratryl alcohol and nonionic surfactant in the oxidation of phenolic compounds by lignin peroxidase

Veratryl alcohol (VA) at higher concentration stimulated the lignin peroxidase (LiP)-catalyzed oxidation of phenolic compounds remarkably. This novel phenomenon was due to its competition with the phenols for the active site of the enzyme and to the high reactivity of the formed cation radical of VA (VA+*) which resulted in an additional oxidation of the phenols. The influence of the nonionic surfactant Tween 80 on the VA-enhanced LiP-catalyzed oxidation of phenols depended on its concentration. At lower concentration it had a small synergetic effect but at higher concentration it decreased the initial rate. Studies of the capillary electrophoretic behavior of LiP in the presence of Tween 80 showed that this effect was caused by the surfactant aggregation on LiP which, at higher surfactant concentrations, might impede the access of VA to its binding site on LiP and, consequently, the VA+* formation.     

Molecular physiology of glutamine and glutamate biosynthesis in developing seedlings of conifers

Nitrogen is a limiting factor in tree growth and development. The incorporation of ammonium ions in carbon skeletons is catalyzed by the sequential action of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT). Most studies on nitrogen‐assimilating enzymes have been reported for annual crop plants. Knowledge of these enzymes in woody plants is much more limited, particularly at the molecular level. Here, we review current available information on glutamine/glutamate biosynthesis and chloroplast development in conifers.     

Assessment of selenium bioavailability from naturally produced high-selenium soy foods in selenium-deficient rats

We assessed the bioavailability of selenium (Se) from a protein isolate and tofu (bean curd) prepared from naturally produced high-Se soybeans. The Se concentrations of the soybeans, the protein isolate and tofu were 5.2 ± 0.2, 11.4 ± 0.1 and 7.4 ± 0.1 mg/kg, respectively. Male weanling Sprague–Dawley rats were depleted of Se by feeding them a 30% Torula yeast-based diet (4.1 μg Se/kg) for 56 days, and then they were replenished with Se for an additional 50 days by feeding them the same diet containing 14, 24 or 30 μg Se/kg from the protein isolate or 13, 23 or 31 μg Se/kg from tofu, respectively. l-Selenomethionine (SeMet) was used as a reference. Selenium bioavailability was determined on the basis of the restoration of Se-dependent enzyme activities and tissue Se concentrations in Se-depleted rats, comparing those responses for the protein isolate and tofu to those for SeMet by using a slope-ratio method. Dietary supplementation with the protein isolate or tofu resulted in linear or log-linear, dose-dependent increases in glutathione peroxidase activities in blood and liver and in thioredoxin reductase activity in liver. Furthermore, supplementation with the protein isolate or tofu resulted in linear or log-linear, dose-dependent increases in the Se concentrations of plasma, liver, muscle and kidneys. These results indicated an overall bioavailability of approximately 101% for Se from the protein isolate and 94% from tofu, relative to SeMet. We conclude that Se from naturally produced high-Se soybeans is highly bioavailable in this model and that high-Se soybeans may be a good dietary source of Se.     

Lateral root development in the maize (Zea mays) lateral rootless1 mutant

Background and Aims

The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions.

Methods

Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data.

Key Results

The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase.

Conclusions

The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no influences observed on the root apical meristem structure and maintenance; however, development of the epidermis and cortex are impaired.