Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.     

Analysis of gammabeta, betagamma, gammagamma, betabeta multiple turns in proteins.

The number of gamma-turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic gamma-turns and 57% inverse gamma-turns are associated as multiple turns with either another y-turn or a beta-turn. We refer to these as multiple turns of the (gammabeta)1,2,3 or (betagamma)1,2,3 type, depending upon whether the gamma-turn is before or after the beta-turn along the protein chain, respectively. However, for multiple turns involving only gamma-turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) beta-turns. Fifty-eight per cent beta-turns are associated as multiple turns with another beta-turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual gamma- or beta-turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side-chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins (http://www.cdfd.org.in/dsmp.html).     

Structural and sequence features of two residue turns in beta‐hairpins

Beta‐turns in beta‐hairpins have been implicated as important sites in protein folding. In particular, two residue β‐turns, the most abundant connecting elements in beta‐hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta‐hairpins with a large data set. For this, 3977 beta‐turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta‐hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two‐residue turns, and their distributions were examined. From this study, we could identify code‐like sequence motifs for the two residue beta‐turn types. Finally, structural and sequence properties of beta‐strands in the beta‐hairpins were analyzed, which revealed that the beta‐strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta‐hairpin turn structures and sequences. Proteins 2014; 82:1721–1733. © 2014 Wiley Periodicals, Inc.     

Analysis and prediction of the different types of beta-turn in proteins

beta-Turns have been extracted from 59 non-identical proteins (resolution 2 A) using the standard criterion that the distance between C alpha (i) and C alpha (i + 3) is less than 7 A (1 A = 0.1 nm). The beta-turns have been classified, using phi, psi angles, into seven conventional turn types (I, I', II, II', IV, VIa, VIb) and a new class of beta-turn, designated type VIII, in which the central residues (i + 1, i + 2) adopt an alpha R beta conformation. Most beta-turn types are found in various topological environments, with the exception of I' and II' beta-turns, where 83% and 50%, respectively, are found in beta-hairpins. Sufficient data have been gathered to enable, for the first time, the separate statistical analysis of type I and II beta-turns. The two turn types have been shown to be strikingly different in their sequence preferences. Type I turns favour Asp, Asn, Ser and Cys at i; Asp, Ser, Thr and Pro at i + 1; Asp, Ser, Asn and Arg at i + 2; Gly, Trp and Met at i + 3, whilst type II turns prefer Pro at i + 1; Gly and Asn at i + 2; Gln and Arg at i + 3. These preferences have been explained by the specific side-chain interactions observed within the X-ray structures. The positional trends for type I and II beta-turns have been incorporated into the simple empirical predictive algorithm originally developed by P.N. Lewis et al. The program has improved the positional prediction of beta-turns, and has enhanced and extended the method by predicting the type of beta-turn. Since the observed preferences reflect local interactions these predictions are applicable not only to proteins, but also to peptides, many of which are thought to contain beta-turns.     

Analysis of gammabeta, betagamma, gammagamma, betabeta continuous turns in proteins.

We report the observation of continuous turns in proteins which comprise individual gamma-turns or beta-turns or both that are situated immediately one after the other along the polypeptide chain. The continuous turns were identified from a representative data set of three-dimensional protein crystal structures. The gammabeta/betagamma, gammagamma and betabeta continuous turns represent peptides of varying amino acid residue lengths and conformations. The continuous turns frequently observed in proteins were: gammabeta, between a coil and a strand; betagamma, between a helix and a strand; gammagamma, between coils; and betabeta, either between a strand and a coil or between strands or coils. We determined the statistically significant amino acid residue preferences at individual positions in the turn, calculated amino acid positional potentials and analyzed main chain hydrogen bonds and side-chain interactions likely to stabilize the continuous turns. The data on continuous turns have been integrated in the database of structural motifs in proteins (DSMP) on our web server at (http://www.cdfd.org.in/dsmp.html). This is useful to make queries on sequences compatible with different continuous turns.     

In silico platform for predicting and initiating β‐turns in a protein at desired locations

Numerous studies have been performed for analysis and prediction of β‐turns in a protein. This study focuses on analyzing, predicting, and designing of β‐turns to understand the preference of amino acids in β‐turn formation. We analyzed around 20,000 PDB chains to understand the preference of residues or pair of residues at different positions in β‐turns. Based on the results, a propensity‐based method has been developed for predicting β‐turns with an accuracy of 82%. We introduced a new approach entitled “Turn level prediction method,” which predicts the complete β‐turn rather than focusing on the residues in a β‐turn. Finally, we developed BetaTPred3, a Random forest based method for predicting β‐turns by utilizing various features of four residues present in β‐turns. The BetaTPred3 achieved an accuracy of 79% with 0.51 MCC that is comparable or better than existing methods on BT426 dataset. Additionally, models were developed to predict β‐turn types with better performance than other methods available in the literature. In order to improve the quality of prediction of turns, we developed prediction models on a large and latest dataset of 6376 nonredundant protein chains. Based on this study, a web server has been developed for prediction of β‐turns and their types in proteins. This web server also predicts minimum number of mutations required to initiate or break a β‐turn in a protein at specified location of a protein. Proteins 2015; 83:910–921. © 2015 Wiley Periodicals, Inc.     

gβ- and gγ-turns in proteins revisited: A new set of amino acid turn-type dependent positional preferences and potentials

The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.     

Water and side‐chain embedded π‐turns

Elucidating protein function from its structure is central to the understanding of cellular mechanisms. This involves deciphering the dependence of local structural motifs on sequence. These structural motifs may be stabilized by direct or water‐mediated hydrogen bonding among the constituent residues. π‐Turns, defined by interactions between (i) and (i + 5) positions, are large enough to contain a central space that can embed a water molecule (or a protein moiety) to form a stable structure. This work is an analysis of such embedded π‐turns using a nonredundant dataset of protein structures. A total of 2965 embedded π‐turns have been identified, as also 281 embedded Schellman motif, a type of π‐turn which occurs at the C‐termini of α‐helices. Embedded π‐turns and Schellman motifs have been classified on the basis of the protein atoms of the terminal turn residues that are linked by the embedded moiety, conformation, residue composition, and compared with the turns that have terminal residues connected by direct hydrogen bonds. Geometrically, the turns have been fitted to a circle and the position of the linker relative to its center analyzed. The hydroxyl group of Ser and Thr, located at (i + 3) position, is the most prominent linker for the side‐chain mediated π‐turns. Consideration of residue conservation among homologous sequences indicates the terminal and the linker positions to be the most conserved. The embedded π‐turn as a binding site (for the linker) is discussed in the context of “nest,” a concave depression that is formed in protein structures with adjacent residues having enantiomeric main‐chain conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 441–453, 2014.     

For public or profession?--The new GMC performance procedures.

The upheaval in the General Medical Council two decades ago came from doctors not the consumers the council was set up to protect. Since then there have been repeated calls for doctors to improve their self regulation by amending the disciplinary procedures. Private member''s bills have failed and the GMC has now proposed performance procedures to deal with doctors who exhibit a "pattern of poor performance." After months of wide consultation in and outside the medical profession the GMC will decide next week whether to endorse the procedures, which unlike the conduct hearings will be inquiries by peers. Professor Margaret Stacey suggests that the procedures lack clarity, smacking of that "trust me" principle whose subtext is "but I''m not telling you what I''m up to."     

Descriptive statistics of disallowed regions and various protein secondary structures in the context of studying twisted β-hairpins

A detailed analysis of polypeptide-chain backbone conformations was carried out for polypeptide-chain segments adjacent to β-turn regions, including the sites of disallowed conformations. A cross comparison of conformations was performed for disallowed regions of the Ramachandran plot and main types of β-turns and adjacent secondary structures. Based on the results, disallowed region 2 (II, II') in the Ramachandran plot was shown to coincide mainly with β-hairpins and, more exactly, twisted β-hairpins. The frequency of residues with angles ?_i, ψ_i that fall in region 2 (II, II') in the latter is 140 times higher than in common β-hairpins.     

Prediction of tight turns and their types in proteins

A tight turn in protein structure is defined as a site where (i) a polypeptide chain reverses its overall direction, i.e., leads the chain to fold back on itself by nearly 180 degrees, and (ii) the amino acid residues directly involved in forming the turn are no more than six. Tight turns are generally categorized as delta-turn, gamma-turn, beta-turn, alpha-turn, and pi-turn, which are formed by two-, three-, four-, five-, and six-amino-acid residues, respectively. According to the folding mode, each of such tight turns can be further classified into several different types. Tight turns play an important role in globular proteins from both the structural and functional points of view. In view of this, various efforts have been made to predict tight turns and their types. This Review summarizes the development in this area, with an emphasis focused on the most recent work concerned that is featured by the sequence-coupled model. Meanwhile, the future challenge in this area has also been briefly addressed.     

Propensities of peptides containing the Asn‐Gly segment to form β‐turn and β‐hairpin structures

The propensities of peptides that contain the Asn‐Gly segment to form β‐turn and β‐hairpin structures were explored using the density functional methods and the implicit solvation model in CH₂Cl₂ and water. The populations of preferred β‐turn structures varied depending on the sequence and solvent polarity. In solution, β‐hairpin structures with βI′ turn motifs were most preferred for the heptapeptides containing the Asn‐Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X‐ray structures. The sequence, H‐bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β‐hairpin structure of each heptapeptide, although the β‐turn segments played a role in promoting the formation of β‐hairpin structures and the β‐hairpin propensity varied. In the heptapeptides containing the Asn‐Gly segment, the β‐hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β‐turns and β‐hairpins containing the Asn‐Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics and the design of binding epitopes for protein–protein and protein–nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653–664, 2016.     

Antibacterial combination therapy using Co3+, Cu2+, Zn2+ and Pd2+ complexes: Their calf thymus DNA binding studies

Four Co(III)-, Cu(II)-, Zn(II)-, and Pd(II)-based potent antibacterial complexes of formula K₃[Co(ox)₃].3H₂O (I), [Cu(bpy)₂Cl]Cl.5H₂O (II), [Zn(bpy)₃]Cl₂ (III), and [Pd(bpy)₂](NO₃)₂ (IV) (where ox is oxalate and bpy is 2,2′-bipyridine) were synthesized. They were characterized by elemental analyses, molar conductance measurements, UV–Vis, FTIR, ¹H NMR, and ¹³C NMR spectra. These metal complexes were ordered in three combination series of I + II, I + II + III, and I + II + III + IV. Antibacterial activity was tested for each of these four metal complexes and their combinations against Gram-positive and Gram-negative bacteria. All compounds were more potent antibacterial agents against the Gram-negative than those of the Gram-positive bacteria. The four metal complexes showed antibacterial activity in the order I > II > III > IV and the activity of their combinations followed the order of I + II + III + IV > I + II + III > I + II. CT-DNA binding studies of complex I and its three combinations were carried out using UV–vis spectral titration, displacement of ethidium bromide (EB), and electrophoretic mobility assay. The results obtained from UV–vis studies indicated that all series interact effectively with CT-DNA. Fluorescence titration revealed that the complexes quench DNA-EB strongly through the static quenching procedures. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) were determined at different temperatures of 293, 300, and 310 K, respectively. The calculated thermodynamic parameters supported that hydrogen binding and Van der Waals forces play a major role in association of each series of metal complexes with CT-DNA and follow the above-binding affinity order for the series.     

WR-1065, an active metabolite of the cytoprotector amifostine, affects phosphorylation of topoisomerase IIα leading to changes in enzyme activity and cell cycle progression in CHO AA8 cells

The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, topoisomerase (topo) IIα activity, and topo IIα phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 μ m of WR-1065 for 30 min did not effect cell cycle progression nor topo IIα activity and phosphorylation status. However, concentrations ranging from 4 μ m to 4 m m were equally effective in significantly altering these three end points. Cell cycle progression was analysed by flow cytometry. Following a 30 min exposure to this range of concentrations, cells redistributed throughout the cell cycle with the most prominent changes being an accumulation of cells in G₂. Topo IIα activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 μ m to 4 m m dose range tested. Likewise, topo IIα phosphorylation levels, analysed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of topo II, decreased between 42% to 48% of control levels. Inhibition of topo IIα activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell cycle progression delay and build-up of cells in the G₂ phase of the cell cycle.     

Does sexual experience affect the strength of male mate choice for high‐quality females in Drosophila melanogaster?

Although females are traditionally thought of as the choosy sex, there is increasing evidence in many species that males will preferentially court or mate with certain females over others when given a choice. In the fruit fly, Drosophila melanogaster, males discriminate between potential mating partners based on a number of female traits, including species, mating history, age, and condition. Interestingly, many of these male preferences are affected by the male''s previous sexual experiences, such that males increase courtship toward types of females that they have previously mated with and decrease courtship toward types of females that have previously rejected them. D. melanogaster males also show courtship and mating preferences for larger females over smaller females, likely because larger females have higher fecundity. It is unknown, however, whether this preference shows behavioral plasticity based on the male''s sexual history as we see for other male preferences. Here, we manipulate the sexual experience of D. melanogaster males and test whether this manipulation has any effect on the strength of male mate choice for large females. We find that sexually inexperienced males have a robust courtship preference for large females that is unaffected by previous experience mating with, or being rejected by, females of differing sizes. Given that female body size is one of the most common targets of male mate choice across insect species, our experiments with D. melanogaster may provide insight into how these preferences develop and evolve.     

Neurexin Ibeta and neuroligin are localized on opposite membranes in mature central synapses

Synaptogenesis requires formation of trans-synaptic complexes between neuronal cell-adhesion receptors. Heterophilic receptor pairs, such as neurexin Iβ and neuroligin, can mediate distinct intracellular signals and form different cytoplasmic scaffolds in the pre- and post-synaptic neuron, and may be particularly important for synaptogenesis. However, the functions of neurexin and neuroligin depend on their distribution in the synapse. Neuroligin has been experimentally assigned to the post-synaptic membrane, while the localization of neurexin remains unclear. To study the subcellular distribution of neurexin Iβ and neuroligin in mature cerebrocortical synapses, we have developed a novel method for the physical separation of junctional membranes and their direct analysis by western blotting. Using urea and dithiothreitol, we disrupted trans-synaptic protein links, without dissolving the lipid phase, and fractionated the pre- and post-synaptic membranes. The purity of these fractions was validated by electron microscopy and western blotting using multiple synaptic markers. A quantitative analysis has confirmed that neuroligin is localized strictly in the post-synaptic membrane. We have also demonstrated that neurexin Iβ is largely (96%) pre-synaptic. Thus, neurexin Iβ and neuroligin normally form trans-synaptic complexes and can transduce bidirectional signals.     

Analysis and design of turns in alpha-helical hairpins

Although the analysis and design of turns that connect the strands in antiparallel beta-hairpins has reached an advanced state, much less is known concerning turns between antiparallel helices in helical hairpins. We have conducted an analysis of the structures and sequence preferences of two types of interhelical turns, each of which connects the two helices by a two-residue linker in an alphaL-beta conformation. Based on this analysis, it became apparent that the turn introduced into a designed four-helix bundle protein, DF1, did not occur within an optimal structural context. DF1 is a dimeric model for the diiron class of proteins. A longer loop with a beta-alphaR-beta conformation was inserted between two helices in the protein, and a sequence was chosen to stabilize its conformation. X-ray crystallography and NMR analysis of the protein showed the structure to be in excellent agreement with design.     

The Effect of Aluminum Exposure on Reproductive Ability in the Bank Vole (<Emphasis Type="Italic">Myodes glareolus</Emphasis>)

Human impact on the environment is steadily increasing the amounts of aluminum in the ecosystems. This element accumulates in plants and water, potentially exposing herbivores to its harmful effect. In heavily polluted sites, a decrease in the density of small rodent populations has been observed. This decline may be caused by many factors, including decreased fertility. The aim of the presented research was to determine how aluminum, administered at concentrations similar to those recorded in industrial districts (Al I = 3 mg/l, Al II = 200 mg/l), affects the reproductive abilities of small rodents. As the indicators of reproductive abilities, body weight, weight of the testes and accessory sex glands of males, and uterus weight of females were estimated. In females, the number of matured follicles (types 6, 7, and 8) was analyzed, while in males, the quantity and quality (matured, viable, swollen, motile, head abnormalities) of epididymal sperm cells were assessed. Moreover, the development of testes, measured by spermatogenic index, was determined. The model species was the bank vole. Our results have proven that aluminum impairs adult individuals’ reproductive abilities by decreasing the quality and quantity of sperm cells and by causing morphologically abnormal development of the gonads. However, no difference in male organometric parameters was found, and only in females treated with 3 mg/l Al, the uterus weight was higher than control. No differences were found in the total number of matured follicles. These results suggest that the decline in rodent numbers in industrial districts is due, at least in part, to poorer males’ reproductive abilities, resulting from exposure to aluminum contamination.