Extracting high-molecular weight DNA from cyanobacteria using Promega's WizardⓇ HMW DNA extraction kit with a modified protocol, METIS.

Extraction of high molecular weight (HMW) DNA for long read sequencing with little to no fragmentation and high purity is difficult to acquire from cyanobacterial species. Here we describe a modified method of extraction using Promega's WizardⓇ HMW DNA Extraction Kit to acquire high molecular weight DNA from two cyanobacterial species. The protocol used in the kit is the "3.D. Isolating HMW DNA from Gram-Positive and Gram-Negative Bacteria" protocol. During a key step in the protocol, we propose that the lingering remnants of the cellular debris such as the mucilage layer of the cyanobacterial species is removed, preventing it from sticking to the DNA pellet produced. This customized modification is done between steps 11 and 12 and called METIS (maximizing extraction, transfer isopropanol step). This step drastically reduces the remaining mucilage layer, which if kept will stick to the DNA and make the DNA unsuitable for sensitive downstream next generation sequencing, like PacBio Sequencing. This protocol has been used to assemble two genomes from cyanobacteria (Synechococcus sp. and Microcystis aeruginosa) and one from a gram-negative bacterium, Lacibacter. It also allows for HMW DNA to be rapidly extracted without the use of toxic chemicals such as phenol and without extra reagents to be purchased.•Maximizing extraction, transfer isopropanol step (METIS) is the key modification during the step of DNA unraveling•METIS reduces leftover remnants of the mucilage layer in the extraction•High molecular weight DNA is produced with little to no fragmentation, and both a high purity and concentration.

Effects of ionic strength on the folding and stability of SAMP1, a ubiquitin-like halophilic protein.

Our knowledge of the folding behavior of proteins from extremophiles is limited at this time. These proteins may more closely resemble the primordial proteins selected in early evolution under extreme conditions. The small archaeal modifier protein 1 (SAMP1) studied in this report is an 87-residue protein with a ß-grasp fold found in the halophile Haloferax volcanii from the Dead Sea. To gain insight into the effects of salt on the stability and folding mechanism of SAMP1, we conducted equilibrium and kinetic folding experiments as a function of sodium chloride concentration. The results revealed that increasing ionic strength accelerates refolding and slows down unfolding of SAMP1, giving rise to a pronounced salt-induced stabilization. With increasing NaCl concentration, the rate of folding observed via a combination of continuous-flow (0.1-2 ms time range) and stopped-flow measurements (>2 ms) exhibited a >100-fold increase between 0.1 and 1.5 M NaCl and leveled off at higher concentrations. Using the Linderström-Lang smeared charge formalism to model electrostatic interactions in ground and transition states encountered during folding, we showed that the observed salt dependence is dominated by Debye-Hückel screening of electrostatic repulsion among numerous negatively charged residues. Comparisons are also drawn with three well-studied mesophilic members of the ß-grasp superfamily: protein G, protein L, and ubiquitin. Interestingly, the folding rate of SAMP1 in 3 M sodium chloride is comparable to that of protein G, ubiquitin, and protein L at lower ionic strength. The results indicate the important role of electrostatic interactions in protein folding and imply that proteins have evolved to minimize unfavorable charge-charge interactions under their specific native conditions.

The nature of persistent interactions in two model ß-grasp proteins reveals the advantage of symmetry in stability.

Two proteins within the ß-grasp superfamily, the B1-domain of protein G and the small archaeal modifier protein 1, were investigated to elucidate the key determinants of structural stability at the level of individual interactions. These symmetrical proteins both contain two ß-hairpins which form a sheet flanked by a central α-helix. They were subjected to high temperature molecular dynamics simulations and the detailed behavior of each long-range interaction was characterized. The results revealed that in GB1 the most stable region was the C-terminal hairpin and in SAMP1 it was the opposite, the N-terminal hairpin. Experimental results for GB1 support this finding. In conclusion, it appears that the difference in the location and number of hydrophobic interactions dictate the differential stability which is accommodated due to structural symmetry of the ß-grasp fold. Thus, the hairpins are interchangeable and in nature this lends itself to adaptability and flexibility.

Survivability of Wild-Type and Genetically Engineered Thermosynechococcus elongatus BP1 with Different Temperature Conditions.

Introduction: Thermosynechococcus elongatus BP1 is a thermophilic strain of cyanobacteria that has an optimum growth at 57°C, and according to previous analysis by Yamaoka et al, T elongatus BP1 cannot survive at a temperature below 30°C. This suggests that the thermophilic property of this strain may be used as a natural biosafety feature to limit the spread of genetically engineered (GE) organisms in the environment if physical containment fails. Objective: To further explore the growth and survivability range of T elongatus BP1, we report a growth and survivability assay of wild-type and GE T elongatus BP1 strains under different conditions. Methods: Wild-type and GE T elongatus BP1 cultures were prepared and incubated in the laboratory (high temperatures and constant light source) and greenhouse conditions (lower/varied temperatures and sunlight) for 4 weeks. The cell density was monitored weekly by measuring the optical density at 730 nm (OD730). To assess the survivability, a sample of each culture was added to fresh media, placed in laboratory conditions (42.2°C and 30 µE m-2 s-1) in multi-well plates and observed for growth for up to three weeks. Lastly, the number of viable cells were determined by plating a diluted sample of the culture on solid media and counting colony-forming units (CFU) after 1 day, 2 weeks and 4 weeks of incubation in laboratory or greenhouse conditions. Results: Our experimental results demonstrated that growth was hindered but that the cells did not entirely die within 2 to 4 weeks at warm temperatures (31.42°C-36.27°C). The study also showed that 2 weeks of exposure to cool temperature conditions (15.44°C-25.30°C) was enough to cause complete death of GE T elongatus BP1. However, it took 2 to 4 weeks for the wild-type T elongatus BP1 cells to die. Conclusion: This study revealed that the thermophilic feature of the T elongatus BP1 may be used as an effective biosafety mechanism at a cool temperature between 15.44°C and 25.30°C but may not be able to serve as a biosafety mechanism at warmer temperatures.

Network Connectivity, Centrality and Fragmentation in the Greek-Key Protein Topology.

Understanding and computationally predicting the protein folding process remains one of the most challenging scientific problems and has uniquely garnered the interdisciplinary efforts of researchers from both the biological, chemical, physical and computational disciplines. Previous studies have demonstrated the importance of long-range interactions in guiding the native structure. However, predicting how the native long-range interaction network forms to generate a specific topology from among all other conformations remains unresolved. The present research study conducts an exploratory study to identify amino acids and long-range interactions that have the potential to play a key role in building and maintaining the protein topology. Towards this end, the application of network science is utilized and developed to analyze the structures of a group of proteins that share a common Greek-key topology but differ in sequence, secondary structure and function. We investigate the idea that the residues with high betweeness centrality score are potentially significant in maintaining the protein network and in governing the Greek-key topology. This hypothesis is tested by two different computational methods: through a fragmentation test and by the analysis of diameter impacts. In summary, we find a subset of selected residues in similar geographical positions in all model proteins, which demonstrates the role of these specific residues and regions in governing the Greek-key topology from a network perspective.

Demonstration of horizontal gene transfer from genetically engineered Thermosynechococcus elongatus BP1 to wild-type E. coli DH5α.

Synthetic biology with genetically engineered (GE) cyanobacteria has the potential to produce valuable products such as biofuels. However, it is also essential to assess the potential risks of synthetic biology technology before it can be widely used. In order to address key concerns posed by the application of synthetic biology to microorganisms, studies were designed to monitor the horizontal transfer of engineered genes from GE cyanobacteria Thermosynechococcus elongatus BP1 to Escherichia coli through co-incubation. The results of these experiments demonstrated that the genetically engineered DNA construct containing alcohol producing genes and kanamycin resistance can be horizontally transferred from GE T. elongatus BP1 to wild-type E. coli following two days of liquid co-culturing. The rapid and facile transfer of foreign genes, which include antibiotic resistance, between bacterial communities signifies the need to continue to deepen our understanding of the process of horizontal gene transfer, chromosomal integration as well as further biosafety-oriented research efforts. In the era of synthetic biology, the natural microbial process for sharing genetic material will also significantly impact risk assessments, containment approaches and further policy development.

Demonstrating significant benefit of orphan medicines: analysis of 15 years of experience in Europe.

In the European Union demonstration of 'significant benefit' is mandatory if satisfactory methods exist for a disease targeted by a new orphan medicinal product. Significant benefit is required at the time of orphan designation, when it can be supported by preclinical studies, and at the time of marketing authorization, when clinical data are needed. For the first time, our work has identified, defined and organized the scientific grounds on which significant benefit is granted in the European Union, based on a review of the orphan medicinal products authorized in the years 2000-2015, and on the working experience of the Committee of Orphan Medicinal Products. The resulting conceptual framework is a tool for medicine developers to reflect on potential areas of advantage of their candidate products, and for a broad range of stakeholders to stimulate the discussion on the added value of orphan medicines across the whole development lifecycle.

Elucidating the Key Determinants of Structure, Folding, and Stability for the ( 4ß+ α ) Conformation of the B1 Domain of Protein G Using Bioinformatics Approaches.

The B1 domain of protein G (GB1) is a small, 56 amino acid bacterial immunoglobulin-binding protein with a 4ß+ α fold. Architecturally, it is composed of a two-layer sandwich consisting of a four-stranded ß -sheet that packs against an α -helix. Using several bioinformatics approaches, we investigated which residues may be key determinants of this fold. We identified nine structurally conserved amino acids using a conservation analysis and propose they are critical to forming and stabilizing the fold. The nine conserved residues form a predominantly hydrophobic nucleus within the core of GB1. A network analysis of all the long-range interactions in the structure of GB1 in concert with a betweenness centrality analysis revealed the relative significance of each conserved amino acid residue based on the number and location of the interactions. This bioinformatics analysis provides an important foundation for the design and interpretation of both computational and experimental work which may be helpful in solving the protein folding problem.

Protein structure networks.

The application of the field of network science to the scientific disciplines of structural biology and biochemistry, have yielded important new insights into the nature and determinants of protein structures, function, dynamics and the folding process. Advancements in further understanding protein relationships through network science have also reshaped the way we view the connectivity of proteins in the protein universe. The canonical hierarchical classification can now be visualized for example, as a protein fold continuum. This review will survey several key advances in the expanding area of research being conducted to study protein structures and folding using network approaches.

Protein folding by 'levels of separation': a hypothesis.

The protein folding process has been studied both computationally and experimentally for over 30 years. To date there is no detailed mechanism to explain the formation of long-range interactions between the transition and native states. Long-range interactions are the principle determinants of the tertiary structure. We present a theoretical model which proposes a mechanism for the acquisition of these interactions as they form in a modified version of 'degrees of separation', that we term 'levels of separation'. It is based on the integration of network science and biochemistry.

Biophysical analysis of the transition of an all α-helical Greek-key protein into amyloid fibrils composed of ß-sheet structure.

Amyloidosis resulting from the deposition of aggregated protein has been linked to many debilitating degenerative diseases which include most notably Alzheimer's and Parkinson's. The tendency for a protein to alternatively form highly ordered amyloid fibrils is dependent on many biological factors. Mutations, temperature, concentration, translational motion and pH play a pivotal role in inducing fibril aggregate assembly in vitro. The key feature appears to be the need to destabilize the native state structure as a required first step. In this paper we report on the detailed conversion of the death domain of the human Fas-associated death domain, an all α-helical protein with a Greek-key topology, into an all ß-sheet amyloid fibril, using a comprehensive range of spectroscopic techniques that provide insight into this process. This transition from α-helical to ß-sheet seems to require destabilization but not complete loss of the secondary structure to explore alternative conformations. This is a fascinating transition that supports the hypothesis that all proteins have the innate ability to form a fibril-like structure. Thus, the primary structure can encode two alternative three-dimensional structures: the native, functional state and the ß-amyloid state. The Fas-associated death domain does not appear to naturally form amyloid fibrils in vivo. Our results clearly indicate that proteins evolved to avoid amyloid fibril formation because we find that the conditions required for formation in our model system are very specific and far from physiological.

Folding of an all-helical Greek-key protein monitored by quenched-flow hydrogen-deuterium exchange and NMR spectroscopy.

To advance our understanding of the protein folding process, we use stopped-flow far-ultraviolet (far-UV) circular dichroism and quenched-flow hydrogen-deuterium exchange coupled with nuclear magnetic resonance (NMR) spectroscopy to monitor the formation of hydrogen-bonded secondary structure in the C-terminal domain of the Fas-associated death domain (Fadd-DD). The death domain superfamily fold consists of six α-helices arranged in a Greek-key topology, which is shared by the all-ß-sheet immunoglobulin and mixed α/ß-plait superfamilies. Fadd-DD is selected as our model death domain protein system because the structure of this protein has been solved by NMR spectroscopy, and both thermodynamic and kinetic analysis indicate it to be a stable, monomeric protein with a rapidly formed hydrophobic core. Stopped-flow far-UV circular dichroism spectroscopy revealed that the folding process was monophasic and the rate is 23.4 s(-1). Twenty-two amide hydrogens in the backbone of the helices and two in the backbone of the loops were monitored, and the folding of all six helices was determined to be monophasic with rate constants between 19 and 22 s(-1). These results indicate that the formation of secondary structure is largely cooperative and concomitant with the hydrophobic collapse. This study also provides unprecedented insight into the formation of secondary structure within the highly populated Greek-key fold more generally. Additional insights are gained by calculating the exchange rates of 23 residues from equilibrium hydrogen-deuterium exchange experiments. The majority of protected amide protons are found on helices 2, 4, and 5, which make up core structural elements of the Greek-key topology.

European regulation on orphan medicinal products: 10 years of experience and future perspectives.

In 2000, regulation on orphan medicinal products was adopted in the European Union with the aim of benefiting patients who suffer from serious, rare conditions for which there is currently no satisfactory treatment. Since then, more than 850 orphan drug designations have been granted by the European Commission based on a positive opinion from the Committee for Orphan Medicinal Products (COMP), and more than 60 orphan drugs have received marketing authorization in Europe. Here, stimulated by the tenth anniversary of the COMP, we reflect on the outcomes and experience gained in the past decade, and contemplate issues for the future, such as catalysing drug development for the large number of rare diseases that still lack effective treatments.

Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding.

BACKGROUND: Chitinases are prevalent in life and are found in species including archaea, bacteria, fungi, plants, and animals. They break down chitin, which is the second most abundant carbohydrate in nature after cellulose. Hence, they are important for maintaining a balance between carbon and nitrogen trapped as insoluble chitin in biomass. Chitinases are classified into two families, 18 and 19 glycoside hydrolases. In addition to a catalytic domain, which is a triosephosphate isomerase barrel, many family 18 chitinases contain another module, i.e., chitinase insertion domain. While numerous studies focus on the biological role of the catalytic domain in chitinase activity, the function of the chitinase insertion domain is not completely understood. Bioinformatics offers an important avenue in which to facilitate understanding the role of residues within the chitinase insertion domain in chitinase function. RESULTS: Twenty-seven chitinase insertion domain sequences, which include four experimentally determined structures and span five kingdoms, were aligned and analyzed using a modified sequence entropy parameter. Thirty-two positions with conserved residues were identified. The role of these conserved residues was explored by conducting a structural analysis of a number of holo-enzymes. Hydrogen bonding and van der Waals calculations revealed a distinct subset of four conserved residues constituting two sequence motifs that interact with oligosaccharides. The other conserved residues may be key to the structure, folding, and stability of this domain. CONCLUSIONS: Sequence and structural studies of the chitinase insertion domains conducted within the framework of evolution identified four conserved residues which clearly interact with the substrates. Furthermore, evolutionary studies propose a link between the appearance of the chitinase insertion domain and the function of family 18 chitinases in the subfamily A.

The CATH hierarchy revisited-structural divergence in domain superfamilies and the continuity of fold space.

This paper explores the structural continuum in CATH and the extent to which superfamilies adopt distinct folds. Although most superfamilies are structurally conserved, in some of the most highly populated superfamilies (4% of all superfamilies) there is considerable structural divergence. While relatives share a similar fold in the evolutionary conserved core, diverse elaborations to this core can result in significant differences in the global structures. Applying similar protocols to examine the extent to which structural overlaps occur between different fold groups, it appears this effect is confined to just a few architectures and is largely due to small, recurring super-secondary motifs (e.g., alphabeta-motifs, alpha-hairpins). Although 24% of superfamilies overlap with superfamilies having different folds, only 14% of nonredundant structures in CATH are involved in overlaps. Nevertheless, the existence of these overlaps suggests that, in some regions of structure space, the fold universe should be seen as more continuous.

Probing the urea dependence of residual structure in denatured human alpha-lactalbumin.

Backbone (15)N relaxation parameters and (15)N-(1)H(N) residual dipolar couplings (RDCs) have been measured for a variant of human alpha-lactalbumin (alpha-LA) in 4, 6, 8 and 10 M urea. In the alpha-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala alpha-LA). This protein is a partially folded molten globule at pH 2 and has been shown previously to unfold in a stepwise non-cooperative manner on the addition of urea. (15)N R(2) values in some regions of all-Ala alpha-LA show significant exchange broadening which is reduced as the urea concentration is increased. Experimental RDC data are compared with RDCs predicted from a statistical coil model and with bulkiness, average area buried upon folding and hydrophobicity profiles in order to identify regions of non-random structure. Residues in the regions corresponding to the B, D and C-terminal 3(10) helices in native alpha-LA show R(2) values and RDC data consistent with some non-random structural propensities even at high urea concentrations. Indeed, for residues 101-106 the residual structure persists in 10 M urea and the RDC data suggest that this might include the formation of a turn-like structure. The data presented here allow a detailed characterization of the non-cooperative unfolding of all-Ala alpha-LA at higher concentrations of denaturant and complement previous studies which focused on structural features of the molten globule which is populated at lower concentrations of denaturant.

Analysis of conservation in the Fas-associated death domain protein and the importance of conserved tryptophans in structure, stability and folding.

Computational and experimental studies focusing on the role of conserved amino acids for folding and stability is an active and promising area of research. To date however, only a small fraction of the potential superfamilies have been investigated. To further expand our understanding we present the results of a bioinformatics analysis of the death domain superfamily. The fold consists of a six helical bundle with a Greek-key topology. Our sequence and structural studies have identified a group of conserved hydrophobic residues and corresponding long-range interactions, which we propose are important in the formation and stabilization of the hydrophobic core and native topology. Six conserved hydrophilic residues were additionally identified and may play a functional role during apoptosis. We also report the establishment of an experimental system that will facilitate studies to test the role of the conserved residues in folding and stability. Equilibrium unfolding and refolding studies of a model superfamily member, Fas-associated death domain protein indicate that the process is cooperative, two-state and reversible. Stopped-flow fluorescence studies reveal that the folding is rapid and biphasic with the majority of the hydrophobic core forming in the first phase. Site-directed mutagenesis studies indicate that conserved tryptophans 112 and 148 are important to structure, native state stability and folding. These results present the earliest conservation analysis and biophysical characterization of the Fas-associated death domain.

The CATH domain structure database: new protocols and classification levels give a more comprehensive resource for exploring evolution.

We report the latest release (version 3.0) of the CATH protein domain database (http://www.cathdb.info). There has been a 20% increase in the number of structural domains classified in CATH, up to 86 151 domains. Release 3.0 comprises 1110 fold groups and 2147 homologous superfamilies. To cope with the increases in diverse structural homologues being determined by the structural genomics initiatives, more sensitive methods have been developed for identifying boundaries in multi-domain proteins and for recognising homologues. The CATH classification update is now being driven by an integrated pipeline that links these automated procedures with validation steps, that have been made easier by the provision of information rich web pages summarising comparison scores and relevant links to external sites for each domain being classified. An analysis of the population of domains in the CATH hierarchy and several domain characteristics are presented for version 3.0. We also report an update of the CATH Dictionary of homologous structures (CATH-DHS) which now contains multiple structural alignments, consensus information and functional annotations for 1459 well populated superfamilies in CATH. CATH is directly linked to the Gene3D database which is a projection of CATH structural data onto approximately 2 million sequences in completed genomes and UniProt.

Characterization of the molten globule of human serum retinol-binding protein using NMR spectroscopy.

The molten globule state is a partially folded conformer of proteins that has been the focus of intense study for more than two decades. This non-native fluctuating conformation has been linked to protein-folding intermediates, to biological function, and more recently to precursors in amyloid fibril formation. The molten globule state of human serum retinol-binding protein (RBP) has been postulated previously to be involved in the mechanism of ligand release (Ptitsyn, O. B., et al. (1993) FEBS Lett. 317, 181-184). Conserved residues within RBP have been identified and proposed to be key to folding and stability, although a link to a molten globule state has not previously been shown (Greene, L. H., et al. (2003) FEBS Lett. 553, 39-44). In this work, a detailed characterization of the acid-induced molten globule of RBP is presented. Using stopped-flow fluorescence spectroscopy in the presence of 8-anilino-1-naphthalene sulfonic acid (ANS), we show that RBP populates a state with molten-globule-like characteristics early in refolding. To gain insight into the structural features of the molten globule of RBP, we have monitored the denaturant-induced unfolding of this ensemble using NMR spectroscopy. The transition at the level of individual residues is significantly more cooperative than that found previously for the archetypal molten globule, alpha-lactalbumin (alpha-LA); this difference may be due to a predominantly beta-sheet structure present in RBP in contrast to the alpha-helical nature of the alpha-LA molten globule.