Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ2, χ3, χ2′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ2 − 60°, χ390, χ2′ − 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.

'Forbidden' disulfides : their role as redox switches

Seminal studies by Richardson and Thornton defined the constraints imposed by protein structure on disulfide formation and flagged forbidden regions of primary or secondary structure seemingly incapable of forming disulfide bonds between resident cysteine pairs. With respect to secondary structure, disulfide bonds were not found between cysteine pairs: A. on adjacent beta-stands; B. in a single helix or strand; C. on non-adjacent strands of the same beta-sheet. In primary structure, disulfide bonds were not found between cysteine pairs: D. adjacent in the sequence. In the intervening years it has become apparent that all these forbidden regions are indeed occupied by disulfide-bonded cysteines, albeit rather strained ones. It has been observed that sources of strain in a protein structure, such as residues in forbidden regions of the Ramachandran plot and cis-peptide bonds, are found in functionally important regions of the protein and warrant further investigation. Like the Ramachandran plot, the earlier studies by Richardson and Thornton have identified a fundamental truth in protein stereochemistry: "forbidden" disulfides adopt strained conformations, but there is likely a functional reason for this. Emerging evidence supports a role for forbidden disulfides in redox-regulation of proteins.

Solvent induced changes in the conformational state of β-lactoglobulin and the influence of protic ionic liquids

The protic ionic liquids (pILs), triethylammonium acetate, triethylammonium trifluoroacetate, triethylammonium mesylate and trimethylammonium sulfate were used to induce various native and non-native conformational states of the protein β-lactoglobulin (βLG). Changes in the secondary structure of βLG were observed on moving from a high water content to a high pIL content. We examined the stability of various pIL induced states via thermal unfolding and refolding, where it was found that at a given pIL concentration a highly stable non-native conformation was formed. The βLG non-native conformation was characterized by a high α-helical content. Additionally, pIL conditions that promoted amyloid fibril formation were identified and characterized by CD, a Thioflavin T binding assay and transmission electron microscopy (TEM). This work highlights the use of pILs as solvents in the study of protein folding using βLG as a model system.

Equilibrium conformational ensemble of the intrinsically disordered peptide n16N: linking subdomain structures and function in nacre

n16 is a framework protein family associated with biogenic mineral stabilization, thought to operate at three key interfaces in nacre: protein/β-chitin, protein/protein, and protein/CaCO3. The N-terminal half of this protein, n16N, is known to be active in conferring this mineral stabilization and organization. While some details relating to the stabilization and organization of the mineral are known, the molecular mechanisms that underpin these processes are not yet established. To provide these molecular-scale details, here we explore current hypotheses regarding the possible subdomain organization of n16N, as related to these three interfaces in nacre, by combining outcomes of Replica Exchange with Solute Tempering molecular dynamics simulations with NMR experiments, to investigate the conformational ensemble of n16N in solution. We verify that n16N lacks a well-defined secondary structure, both with and without the presence of Ca(2+) ions, as identified from previous experiments. Our data support the presence of three different, functional subdomains within n16N. Our results reveal that tyrosine, chiefly located in the center of the peptide, plays a multifunctional role in stabilizing conformations of n16N, for intrapeptide and possibly interpeptide interactions. Complementary NMR spectroscopy data confirm the participation of tyrosine in this stabilization. The C-terminal half of n16N, lacking in tyrosine and highly charged, shows substantive conformational diversity and is proposed as a likely site for nucleation of calcium carbonate. Finally, dominant structures from our predicted conformational ensemble suggest the presentation of key residues thought to be critical to the selective binding to β-chitin surfaces.

Drying and denaturation characteristics of α-lactalbumin, β-lactoglobulin, and bovine serum albumin in a convective drying process

Drying and denaturation kinetics of aqueous droplets of α-lactalbumin (α-lac), β-lactoglobulin (β-lg), and bovine serum albumin (BSA) were measured in a convective drying environment. Single droplets having an initial droplet diameter of 2 ± 0.1 mm and containing 10% (w/v) protein concentration were dried using conditioned air (65 and 80 °C, 2-3% RH, 0.5 m/s velocity) for 600 s. The denaturation of these proteins was measured by using reversed-phase HPLC. At the end of 600 s of drying 13.3 and 19.4% α-lac was found to be lost due to denaturation at 65 and 80 °C, respectively. Up to 31.0% of β-lg was found to be denatured, whereas BSA was not found to be significantly (p > 0.05) denatured in these drying conditions. The formation and strength of skin and the associated morphological features were found to be linked with the degree of denaturation of these proteins. The secondary structure of these proteins was significantly (p < 0.05) affected and altered by the drying stresses. The β-sheet and random coil contents were increased in α-lac by 6.5 and 4.0%, respectively, whereas the α-helix and β-turn contents decreased by 5.5 and 5.0%, respectively. The β-sheet and random coil contents in β-lg were increased by 7.5 and 2.0%, respectively, whereas the α-helix and β-turn contents decreased by 3.5 and 6.0%, respectively. In the case of BSA the β-sheet, α-helix, and random coil contents were found to increase, whereas the β-turn content decreased.

Testing the transferability of a coarse-grained model to intrinsically disordered proteins

The intermediate-resolution coarse-grained protein model PLUM [T. Bereau and M. Deserno, J. Chem. Phys., 2009, 130, 235106] is used to simulate small systems of intrinsically disordered proteins involved in biomineralisation. With minor adjustments to reduce bias toward stable secondary structure, the model generates conformational ensembles conforming to structural predictions from atomistic simulation. Without additional structural information as input, the model distinguishes regions of the chain by predicted degree of disorder, manifestation of structure, and involvement in chain dimerisation. The model is also able to distinguish dimerisation behaviour between one intrinsically disordered peptide and a closely related mutant. We contrast this against the poor ability of PLUM to model the S1 quartz-binding peptide.

Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer

Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzinostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the β sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corroborate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic modalities towards targeted cancer therapy.

Predicting molecular structures: an application of the cutting angle method

The ability to predict molecular geometries has important applications in chemistry. Specific examples include the areas of protein space structure elucidation, the investigation of host–guest interactions, the understanding of properties of superconductors and of zeolites. This prediction of molecular geometries often depends on finding the global minimum or maximum of a function such as the potential energy. In this paper, we consider several well-known molecular conformation problems to which we apply a new method of deterministic global optimization called the cutting angle method. We demonstrate that this method is competitive with other global optimization techniques for these molecular conformation problems.

Study of the natriuretic peptide hormone system in plants

In this study, both physiological and cellular effects are elicited by natriuretic peptides (NPs), a novel type of plant hormone. It was found that rat ANP (rANP) influenced stomatal opening movement in Tradescantia sp., where a significant increase in stomatal opening was observed in the presence of 1 µM rANP. Furthermore, this effect is mediated by cGMP, a (putative) second messenger of NPs. Two inhibitors of guanylyl cyclase, LY 83583 and methylene blue, inhibited rANP-induced stomatal opening. In contrast, stomatal opening is induced in a concentration dependent manner by the cell permeant cGMP analogue 8-Br-cGMP. In addition it was found, that like in animals, the secondary structure of rANP is essential for rANP responses. Linearised rANP is biologically inactive. Since ANP elicit plant responses, an attempt was made to isolate NP analogues from plants. A protocol for partially purifying NP from plants was developed. It was found that two fractions eluted from an immunoaffinity chromatography column (0.5 M KCI eluted fraction and 0.75 M KCI eluted fraction) were biologically active. The level of cGMP in response to NPs was also tested. It is suggested that the receptor of NP is specific since only 0.75 M KCI eluted fractions increased cGMP levels in Zea mays root stele tissue. rANP did not elicit an effect on cGMP levels in this tissue and LY 83583 did not affect this response. It is therefore argued that a plant specific biologically active NP system is present in the stele and it is predicted that NPs modulate solute movement in this tissue. NPs also influence K⁺, Na⁺ and H⁺ fluxes in Zea mays root stele. Increase in both K⁺ and Na+ uptake were observed after 30 min., while H⁺ flux shifted immediately toward influx in the presence of both 0.5 and 0.75 KCI eluted fractions. Finally, a model is proposed for the effect of NPs on solute movement and its signalling system in plants.

Superhydrophobic electrospun POSS-PMMA copolymer fibres with highly ordered nanofibrillar and surface structures

A POSS-PMMA copolymer has been synthesised by conventional free-radical polymerisation reaction. Uniform electrospun fibres from this copolymer showed a water contact angle as high as 1651 with a sliding angle as low as 61. For the first time, we found that the electrospun fibres had a bundled nanofibril secondary structure with an ordered POSS morphology on the fibre surface.

High torsional energy disulfides : relationship between cross-strand disulfides and right-handed staples

Redox-active disulfides are capable of being oxidized and reduced under physiological conditions. The enzymatic role of redox-active disulfides in thiol-disulfide reductases is well-known, but redox-active disulfides are also present in non-enzymatic protein structures where they may act as switches of protein function. Here, we examine disulfides linking adjacent β-strands (cross-strand disulfides), which have been reported to be redox-active. Our previous work has established that these cross-strand disulfides have high torsional energies, a quantity likely to be related to the ease with which the disulfide is reduced. We examine the relationship between conformations of disulfides and their location in protein secondary structures. By identifying the overlap between cross-strand disulfides and various conformations, we wish to address whether the high torsional energy of a cross-strand disulfide is sufficient to confer redox activity or whether other factors, such as the presence of the cross-strand disulfide in a strained β-sheet, are required.

Nerve guidance conduits from aligned nanofibers: improvement of nerve regeneration through longitudinal nanogrooves on a fiber surface

A novel fibrous conduit consisting of well-aligned nanofibers with longitudinal nanogrooves on the fiber surface was prepared by electrospinning and was subjected to an in vivo nerve regeneration study on rats using a sciatic nerve injury model. For comparison, a fibrous conduit having a similar fiber alignment structure without surface groove and an autograft were also conducted in the same test. The electrophysiological, walking track, gastrocnemius muscle, triple-immunofluorescence, and immunohistological analyses indicated that grooved fibers effectively improved sciatic nerve regeneration. This is mainly attributed to the highly ordered secondary structure formed by surface grooves and an increase in the specific surface area. Fibrous conduits made of longitudinally aligned nanofibers with longitudinal nanogrooves on the fiber surface may offer a new nerve guidance conduit for peripheral nerve repair and regeneration.

RNASeqBrowser: a genome browser for simultaneous visualization of raw strand specific RNAseq reads and UCSC genome browser custom tracks

BACKGROUND: Strand specific RNAseq data is now more common in RNAseq projects. Visualizing RNAseq data has become an important matter in Analysis of sequencing data. The most widely used visualization tool is the UCSC genome browser that introduced the custom track concept that enabled researchers to simultaneously visualize gene expression at a particular locus from multiple experiments. Our objective of the software tool is to provide friendly interface for visualization of RNAseq datasets.

RESULTS: This paper introduces a visualization tool (RNASeqBrowser) that incorporates and extends the functionality of the UCSC genome browser. For example, RNASeqBrowser simultaneously displays read coverage, SNPs, InDels and raw read tracks with other BED and wiggle tracks -- all being dynamically built from the BAM file. Paired reads are also connected in the browser to enable easier identification of novel exon/intron borders and chimaeric transcripts. Strand specific RNAseq data is also supported by RNASeqBrowser that displays reads above (positive strand transcript) or below (negative strand transcripts) a central line. Finally, RNASeqBrowser was designed for ease of use for users with few bioinformatic skills, and incorporates the features of many genome browsers into one platform.

CONCLUSIONS: The features of RNASeqBrowser: (1) RNASeqBrowser integrates UCSC genome browser and NGS visualization tools such as IGV. It extends the functionality of the UCSC genome browser by adding several new types of tracks to show NGS data such as individual raw reads, SNPs and InDels. (2) RNASeqBrowser can dynamically generate RNA secondary structure. It is useful for identifying non-coding RNA such as miRNA. (3) Overlaying NGS wiggle data is helpful in displaying differential expression and is simple to implement in RNASeqBrowser. (4) NGS data accumulates a lot of raw reads. Thus, RNASeqBrowser collapses exact duplicate reads to reduce visualization space. Normal PC's can show many windows of NGS individual raw reads without much delay. (5) Multiple popup windows of individual raw reads provide users with more viewing space. This avoids existing approaches (such as IGV) which squeeze all raw reads into one window. This will be helpful for visualizing multiple datasets simultaneously. RNASeqBrowser and its manual are freely available at http://www.australianprostatecentre.org/research/software/rnaseqbrowser or http://sourceforge.net/projects/rnaseqbrowser/.

Development and evaluation of a novel insulin analogue

 This project examined the impact of pegylation on secondary structure of insulin during tablet making process and assessed the metabolism in in-vitro and in-vivo model. It was found that pegylation per se does not affect the secondary structure of insulin. The tablet making process did not have any impact on the pegylated insulin structure. In the in vitro model and in vivo model, pegylated lysine and free peg molecule were found to be the metabolites specific to pegylated insulin.

Targeting cancer cells using LNA-modified aptamer-siRNA chimeras

Aptamers are chimerized with drug or antisense oligos or nanoparticles to generate targeted therapeutics for cancer. Aptamer chimerized siRNA rescues nonspecific delivery and, thereby, enhances the availability of siRNA to target cells. EpCAM RNA aptamer (EpApt or Ep) has potential for siRNA chimerization due to its secondary structure. Stathmin and survivin proteins are reported to aid oncogenicity in retinoblastoma (RB), breast cancer and other cancers. Thus, chimerization of EpCAM Apt with siRNA against survivin and stathmin, respectively, was performed by incorporating Locked Nucleic Acid (LNA) modification. The LNA-modified chimeric aptamers were stable until 96 h and got internalized into RB, WERI-Rb1 and breast cancer, MDAMB453 cell lines. The constructs were studied using the recombinant dicer enzyme for the siRNA generation. Quantitative polymerase chain reaction and immunofluorescence by microscopic analysis of chimeras in vitro exhibited silencing of stathmin and survivin in the RB and breast cancer model. The chimeric constructs showed significant inhibition of cell proliferation of breast cancer cells. Thus, LNA-modified aptamer-based siRNA delivery aids in cell targeting and necessitates further studies in animal models.