Interresidue interactions in protein classes

The free energy difference between folded and unfolded state is about the same for most proteins and it is not more than the energy of a few noncovalent interactions. In addition to the numerous noncovalent interactions, some proteins contain one or more disulfide bonds, which, as covalent crosslinks, significantly stabilize their tertiary structure. Correlation between the presence of disulfide bond(s), and the number noncovalent interresidue interactions of various kinds is analyzed here. The number of interactions per residue is almost the same for all protein. Also the number of long-range interactions per residue is the same in all proteins. Proteins with S(SINGLE BOND)S bond(s) (extracellular proteins) have more medium-range and fewer short-range interactions than those without S(SINGLE BOND)S bonds. However, the difference is independent of the number of these covalent crosslinks. We concluded that the different distributions of the various kinds of noncovalent interaction reflect the needs of proteins in the different environments, the extracellular and the intracellular ones, rather than the presence of the disulfide bond(s). We also pointed out that the observed differences in the distributions of short- and medium-range interactions are in good agreement with different secondary structure compositions of extracellular and intracellular proteins. Proteins 27:360–366, 1997. © 1997 Wiley-Liss, Inc.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

Alterations in the sugar metabolism and in the vacuolar system of mesophyll cells contribute to the desiccation tolerance of <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis> ecotypes

Haberlea rhodopensis belongs to the small group of resurrection plants having the unique ability to survive desiccation to air dry state retaining most of its chlorophyll content and then resume normal function upon rehydration. It prefers the shady valleys and northward facing slopes of limestone ridges in mountain zones with high average humidity. Nevertheless, it can be found rarely on rocks directly exposed to the sunlight, without the coverage of the canopy. In the present study, we follow the alterations in the subcellular organization of mesophyll cells and sugar metabolism upon desiccation of shade and sun H. rhodopensis plants. Composition and content of soluble carbohydrates during desiccation and rehydration were different in plants grown below the trees or on the sunny rocks. Sucrose, however, was dominating in both ecotypes. The amount of starch grains in chloroplasts was inversely related to that of sugars. Concomitantly with these changes, the number of vacuoles was multiplied in the cells. This can be explained by the development of small (secondary) vacuoles peripherally in the cytoplasm, rather than by the fragmentation of the single vacuole, proposed earlier in the literature. Accordingly, the centripetal movement of chloroplasts and other organelles may be a result of the dynamic changes in the vacuolar system. Upon rehydration, the inner vacuoles enlarged and the organelles returned to their normal position.     

13C NMR snapshots of the complex reaction coordinate of pyridoxal phosphate synthase

The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate.     

Adjustments of water use efficiency by stomatal regulation during drought and recovery in the drought-adapted Vitis hybrid Richter-110 (V. berlandieri x V. rupestris)

The hybrid Richter-110 (Vitis berlandieri x Vitis rupestris) (R-110) has the reputation of being a genotype strongly adapted to drought. A study was performed with plants of R-110 subjected to water withholding followed by re-watering. The goal was to analyze how stomatal conductance (g(s)) is regulated with respect to different physiological variables under water stress and recovery, as well as how water stress affects adjustments of water use efficiency (WUE) at the leaf level. Water stress induced a substantial stomatal closure and an increase in WUE, which persisted many days after re-watering. The g(s) during water stress was mainly related to the content of ABA in the xylem and partly related to plant hydraulic conductivity but not to leaf water potential. By contrast, low g(s) during re-watering did not correlate with ABA contents and was only related to a sustained decreased hydraulic conductivity. In addition to a complex physiological regulation of stomatal closure, g(s) and rate of transpiration (E) were strongly affected by leaf-to-air vapor pressure deficit (VPD) in a way dependent of the treatment. Interestingly, E increased with increasing VPD in control plants, but decreased with increasing VPD in severely stressed plants. All together, the fine stomatal regulation in R-110 resulted in very high WUE at the leaf level. This genotype is revealed to be very interesting for further studies on the physiological mechanisms leading to regulation of stomatal responsiveness and WUE in response to drought.     

Effects of drought on water content and photosynthetic parameters in potato plants expressing the trehalose-6-phosphate synthase gene of Saccharomyces cerevisiae

Two transgenic potato lines, T1 and T2, expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast were isolated. In our experimental approach, we applied two novelties, namely the fusion of the drought-inducible promoter StDS2 to TPS1 and a marker-free transformation method. In contrast to the expected drought-induced expression, only a very low constitutive TPS1 expression was detected in the transgenic lines, probably due to chromosomal position effects. The observed expression pattern, however, was sufficient to alter the drought response of plants. Detached leaves of T1 and T2 showed an 8 h delay in wilting compared to the non-transformed control. Potted plants of T1 and T2 kept water 6 days longer than control plants and maintained high stomatal conductance and a satisfactory rate of net photosynthesis. During drought treatment, CO₂ assimilation rate measured at saturating CO₂ level was maintained at maximum level for 6–9 days in transgenic plants while it decreased rapidly after 3 days in the wild type plants. Under optimal growth conditions, lower CO₂ fixation was detected in the transgenic than in the control plants. Stomatal densities of T1 and T2 leaves were reduced by 30–40%. This may have contributed to the lower CO₂ fixation rate and altered drought response. Ibolya Stiller, and Sándor Dulai contributed equally to this work.     

Old nests are cues for suitable breeding sites in the Eurasian penduline tit Remiz pendulinus

Nest-sites often have a major influence on avian reproductive success. The use of reliable cues that assist nest-site selection should thus be favoured by natural selection. The old nests have been known to serve as a cue in nest-site selection in several species. To find out whether the old nests act as cue in nest-site selection in the Eurasian penduline tit Remiz pendulinus , we carried out two experiments in southern Hungary, where the penduline tits breed around fishponds and build sophisticated pendulous nests on tree branches that often hang over water. In April 2006, we choose 20 groups of two nearby trees, and hung an old nest on one of the trees in each group. The male penduline tits choose 12 of these groups to build a new nest, and every of the twelve nests were built on trees with an old nest. This suggests that the old nests serve as a cue in the selection of breeding sites for males when they enter a habitat. To find out whether the old nests are cues of plentiful nest building material, or to signal high quality breeding areas, we carried out a second experiment in 2007 by selecting 13 groups of three nearby trees. A "worn-out" old nest was hung on one of the trees, a "re-utilize" type of old nest on another tree, whereas the third tree was left without an old nest in each group. The rationale was that while the worn-out material of the old nests is of no use in building the new nests, material of the "re-utilize" nests is good enough to be used for building new nests. Males built a new nest in 10 of the 13 groups, and eight of the new nests were built on trees with an old nest. Of the eight new nests, five were built on trees with a "worn-out" old nest and three on trees with a "re-utilize" type of old nest. It appears that for the penduline tit males it is the presence of an existing old nest and not the quality of the old nest material that serves as cue during the selection of the suitable breeding sites.     

Abscisic acid and auxin accumulation in Catasetum fimbriatum roots growing in vitro with high sucrose and mannitol content

Endogenous contents of indolyl-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in excised roots of Catasetum fimbriatum (Orchidaceae) cultured in vitro on solidified Vacin and Went medium with 1, 2, 4, 6, 8 and 10 % sucrose, as well as 2 % sucrose plus mannitol. Maximum root growth was observed in media with 4 % sucrose and 2 % sucrose plus 2.2 % mannitol, suggesting that a moderate water or osmotic stress promotes orchid root growth. Contents of both ABA and IAA increased in parallel to increasing sucrose concentration and a correlation between root elongation and the ABA/IAA ratio was observed. Incubating isolated C. fimbriatum roots with radiolabeled tryptophan, we showed an accumulation of IAA and its conjugates.     

Generation of Marker- and Backbone-Free Transgenic Potatoes by Site-Specific Recombination and a Bi-Functional Marker Gene in a Non-Regular One-Border Agrobacterium Transformation Vector

A binary vector, designated PROGMO, was constructed to assess the potential of the Zygosaccharomyces rouxii R/Rs recombination system for generating marker- and backbone-free transgenic potato (Solanum tuberosum) plants with high transgene expression and low copy number insertion. The PROGMO vector utilises a constitutively expressed plant-adapted R recombinase and a codA-nptII bi-functional, positive/negative selectable marker gene. It carries only the right border (RB) of T-DNA and consequently the whole plasmid will be inserted as one long T-DNA into the plant genome. The recognition sites (Rs) are located at such positions that recombinase enzyme activity will recombine and delete both the bi-functional marker genes as well as the backbone of the binary vector, leaving only the gene of interest flanked by a copy of Rs␣and RB. Efficiency of PROGMO transformation was tested by introduction of the GUS reporter gene into potato. It was shown that after 21 days of positive selection and using 300 mgl⁻¹ 5-fluorocytosine for negative selection, 29% of regenerated shoots carried only the GUS gene flanked by a copy of Rs and RB. The PROGMO vector approach is simple and might be widely applicable for the production of marker- and backbone-free transgenic plants of many crop species.