The optimal fraction of hydrophobic residues required to ensure protein collapse

The hydrophobic interaction is the main driving force for protein folding. Here, we address the question of what is the optimal fraction, f of hydrophobic (H) residues required to ensure protein collapse. For very small f (say f<0.1), the protein chain is expected to behave as a random coil, where the H residues are "wrapped" locally by polar (P) residues. However, for large enough f this local coverage cannot be achieved and the thermodynamic alternative to avoid contact with water is burying the H residues in the interior of a compact chain structure. The interior also contains P residues that are known to be clustered to optimize their electrostatic interactions. This means that the H residues are clustered as well, i.e. they effectively attract each other like the H-monomers in Dill's HP lattice model. Previously, we asked the question: assuming that the H monomers in the HP model are distributed randomly along the chain, what fraction of them is required to ensure a compact ground state? We claimed there that f approximately p(c), where p(c) is the site percolation threshold of the lattice (in a percolation experiment, each site of an initially empty lattice is visited and a particle is placed there with a probability p. The interest is in the critical (minimal) value, p(c), for which percolation occurs, i.e. a cluster connecting the opposite sides of the lattice is created). Due to the above correspondence between the HP model and real proteins (and assuming that the H residues are distributed at random) we suggest that the experimental f should lead to percolating clusters of H residues over the highly dense protein core, i.e. clusters of the core size. To check this theory, we treat a simplified model consisting of H and P residues represented by their alpha-carbon atoms only. The structure is defined by the C(alpha)-C(alpha) virtual bond lengths, angles and dihedral angles, and the X-ray structure is best-fitted onto a face-centered cubic lattice. Percolation experiments are carried out for 103 single-chain proteins using six different hydrophobic sets of residues. Indeed, on average, percolating clusters are generated, which supports our theory; however, some sets lead to a better core coverage than others. We also calculate the largest actual hydrophobic cluster of each protein and show that, on average, these clusters span the core, again in accord with our theory. We discuss the effect of protein size, deviations from the average picture, and implications of this study for defining reliable simplified models of proteins.     

Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy

The four-way DNA (Holliday) junction is an essential intermediate in DNA recombination, and its dynamic characteristics are likely to be important in its cellular processing. In our previous study we observed transitions between two antiparallel stacked conformations using a single-molecule fluorescence approach. The magnesium concentration-dependent rates of transitions between stacking conformers suggested that an unstacked open structure, which is stable in the absence of metal ions, is an intermediate. Here, we sought to detect possible rare species such as open and parallel conformations and further characterized ionic effects. The hypothesized open intermediate cannot be resolved directly due to the limited time resolution and sensitivity, but our study suggests that the open form is achieved very frequently, hundreds of times per second under physiologically relevant conditions. Therefore despite being a minority species, its frequent formation raises the probability that it could become stabilized by protein binding. By contrast, we cannot detect even a transient existence of the junctions in a parallel form, and the probability of such forms with a lifetime greater than 5 ms is less than 0.01%. Stacking conformer transitions are observable in the presence of sodium or hexammine cobalt (III) ions as well as magnesium ions, but the transition rates are higher for lower valence ions at the same concentrations. This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms.     

Chloroperoxidase from <Emphasis Type="Italic">Caldariomyces fumago</Emphasis> is active in the presence of an ionic liquid as co-solvent

Chloroperoxidase from Caldariomyces fumago catalyses the oxidation of 1,2-dihydronaphthalene to (1R,2R)-(+)-dihydroxytetrahydronaphthalene in homogenous citrate buffer/ionic liquid mixtures, using t-butyl hydroperoxide as O<>₂<> donor. It tolerates up to 30 (v/v) 1,3-dimethylimidazolium methylsulfate or 1-butyl-3-methylimidazolium methylsulfate. The enzyme activity in these ionic liquid co-solvent systems is retained for 24h, but it falls to 3h using non-ionic organic solvents such as t-BuOH or acetone.     

Apoptotic volume decrease, pH acidification and chloride channel activation during apoptosis requires CD45 expression in HPB-ALL T cells

Mitochondrial-perturbating agents such as toxic coumponds induce apoptosis. We note that the loss of CD45 expression in the lymphoblastic leukemia cell line HPB-ALL (HPB45.0) leads to an inhibition of nuclear apoptosis. Our hypothesis is that the absence of CD45 disturbs protein function regulated by a proto-oncogene of the Src family playing a significant role in nuclear apoptosis. In this work we explore the importance of a chloride efflux on DNA fragmentation. The role of tyrosine kinase in the function and regulation of the chloride channels was determined. Our results showed a disturbance of ionic homeostasis in CD45 deficient lymphocytes (CD45-) in contrast to normal lymphocytes (CD45+). The phosphorylation levels of the chloride channels are considerably inhibited in CD45-, while the expression levels of these channels are similar in the two types of cells. A hypertonic medium inhibits DNA fragmentation in CD45+ while a hypotonic medium increases DNA fragmentation in CD45-. Thus CD45 plays a significant role in nuclear apoptosis by the regulation of the chloride channels responsible for ionic homeostasis of the cell essential for the DFF40 activation.     

Circularly polarized chlorophyll luminescence reflects the macro-organization of grana in pea chloroplasts

Circular polarization of luminescence (CPL; Steinberg IZ (1978) Annu Rev Biophys Bioeng 7: 113–137) was applied to study pea chloroplasts in different structural states. The structural changes of chloroplasts were induced by variation of osmotic pressure, concentration of magnesium-ions or photoinhibition. Both large CPL and psi-type circular dichroism (psi, polymerization and salt induced) signals appeared in the presence of granal macrostructure and were sensitive to structural changes of the grana. The relation was studied between the amount of CPL expressed as an emission anisotropy factor g _em and amplitudes of the red psi-type CD bands. The positive psi-type CD band was not directly correlated with g _em possibly due to a large contribution of circular intensity differential scattering to the measured CD spectra. However, a linear correlation between the amplitude of the negative psi-type CD band and g _em was found. The CPL signal of pea chloroplasts was attributed to a psi-type origin, which is observed in macroaggregates with densely packed chromophores with a long-range chiral order, and directly depends on the level of macroorganization. With the use of CPL-based microscopy, the long-range packing of LHC II particles can be studied in individual chloroplasts in future. In addition, the CPL method in general allows the study of the macro-organization of grana in green leaves, where conventional light-transmission methods fail. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alpha-chymotrypsin catalysis in imidazolium-based ionic liquids

The transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with 1-propanol catalyzed by alpha-chymotrypsin was examined in the ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim][PF(6)]) and 1-octyl-3-methylimidazolium hexafluorophosphate ([omim][PF(6)]), and in combination with supercritical carbon dioxide (SC-CO(2)). The activity of alpha-chymotrypsin was studied to determine whether trends in solvent polarity, water activity, and enzyme support properties, observed with this enzyme in conventional organic solvents, hold for the novel environment provided by ionic liquids. alpha-Chymotrypsin freeze-dried with K(2)HPO(4), KCl, or poly(ethylene glycol) demonstrated no activity in [bmim][PF(6)] or [omim][PF(6)] at very low water concentrations, but moderate transesterification rates were observed with the ionic liquids containing 0.25% water (v/v) and higher. However, the physical complexation of the enzyme with poly(ethylene glycol) or KCl did not substantially stimulate activity in the ionic liquids, unlike that observed in hexane or isooctane. Activities were considerably higher in [omim][PF(6)] than [bmim][PF(6)]. Added water was not necessary for enzyme activity when ionic liquids were combined with SC-CO(2). These results indicate that [bmim][PF(6)] and [omim][PF(6)] provide a relatively polar environment, which can be modified with nonpolar SC-CO(2) to optimize enzyme activity.     

Conformational behavior of ionic self-complementary peptides

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable beta-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the alpha-helical dipole moment (C --> N), resulting in a strong tendency to form stable alpha-helices as well. Therefore, these peptides can form both stable alpha-helices and beta-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable beta-sheet and alpha-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.     

Influence of Short-Chain Aliphatic Acids on the Phenanthrene Desorption and Mobilization from Contaminated Soil

This study was performed to investigate the influence of short-chain aliphatic acids (SCAAs) on the desorption of phenanthrene from artificially contaminated soils with this polycyclic aromatic hydrocarbon. Five SCAAs examined, including acetic acid, oxalic acid, malic acid, tartaric acid and citric acid, were related to the increase of phenanthrene desorption from two kinds of soil. Citric acid and oxalic acid enhanced phenanthrene desorption to a more significant extent than other organic acids. The effects of pH, SCAA concentration, and ionic strength were further evaluated. The phenanthrene desorption was enhanced as the pH increased. An increase in desorbed phenanthrene from pH 3 to pH 8 was observed, but that was followed by a slight decrease above pH 8 for most SCAAs. The phenanthrene desorption performance showed increments with increasing organic acid concentrations. However, the increase of phenanthrene desorption became less remarkable when SCAA concentrations were above 100 mmol/L. Moreover the results suggested that high ionic strength hindered the desorption of phenanthrene in the presence of SCAAs.     

Calcium and salinity as selective factors in plate morph evolution of the three-spined stickleback (Gasterosteus aculeatus)

Identifying the causal factors underlying natural selection remains a key challenge in evolutionary biology. Although the genetic basis for the plate morph evolution of three-spined stickleback (Gasterosteus aculeatus) is well described, the environmental variables that form the basis for different morphs are not understood. We measured the effects of dissolved calcium and salinity on the growth of sticklebacks with different plate morphs from Scotland and Poland. There was a significant interaction of calcium with plate morph for fish from both regions, with complete morph sticklebacks growing more slowly at low calcium concentrations and low morph sticklebacks showing divergent responses to calcium concentration. A Scottish anadromous population showed evidence of local adaptation to high salinity, which was independent of plate morph. Polish and Scottish populations diverged in their response to salinity, suggesting a difference in osmotic regulation. The results implicate a role for calcium in selecting for plate morph evolution in sticklebacks, possibly as a limiting element in skeletal growth.