Search

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.

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.

The B1 domain of protein G (GB1) is a small, 56 amino acid bacterial immunoglobulin-binding protein with a 4β+ α fold. Architecturally, it is composed of a two-layer sandwich consisting of a four-stranded β -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.

Backbone 15N relaxation parameters and 15N–1HN residual dipolar couplings (RDCs) have been measured for a variant of human α-lactalbumin (α-LA) in 4, 6, 8 and 10 M urea. In the α-LA variant, the eight cysteine residues in the protein have been replaced by alanines (all-Ala α-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. 15N R2 values in some regions of all-Ala α-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 310 helices in native α-LA show R2 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 α-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. Electronic supplementary material The online version of this article (doi:10.1007/s10858-009-9342-y) contains supplementary material, which is available to authorized users.

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.

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.

Investigations into the nature of sequence and structural conservation underlying protein folds have recently yielded profound insights into the mechanism of protein folding and stability. Combining this avenue of research with principles being pioneered in the field of network science holds the promise to further extend the boundaries of our knowledge. In this report we propose that critical determinants of a protein's native topology are encoded by a conserved network of interactions between amino acids from geographically important positions. This hypothesis is based on the novel elucidation of a conserved network of long-range interactions within a set of proteins that share a Greek-key topology and similar chain length, but differ in secondary structure composition, function and sequence. Exploratory macromolecular simulations using the conserved networks as constraints were successful in generating the gross native-like topology from a random linear coil for each of the model proteins. The results indicate that the conserved network contains governing features and supports the idea that the geographical location of these residue interactions is a pivotal feature underlying their conservation. The partially folded model proteins also display a clear scale-free distribution of long-range interactions. To further test the hypothesis, the network parameter betweeness-centrality was calculated for the protein structure networks of our model proteins and highlights two structural elements as particularly vital to the structural stability of the network topology.

Traditionally, proteins have been viewed as a construct based on elements of secondary structure and their arrangement in three-dimensional space. In a departure from this perspective we show that protein structures can be modelled as network systems that exhibit small-world, single-scale, and to some degree, scale-free properties. The phenomenological network concept of degrees of separation is applied to three-dimensional protein structure networks and reveals how amino acid residues can be connected to each other within six degrees of separation. This work also illuminates the unique features of protein networks in comparison to other networks currently studied. Recognising that proteins are networks provides a means of rationalising the robustness in the overall three-dimensional fold of a protein against random mutations and suggests an alternative avenue to investigate the determinants of protein structure, function and folding.

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

alpha-Lactalbumins and the type-c lysozymes are homologues with similar folds that differ in function and stability. To determine if the lower stability of alpha-lactalbumin results from specific substitutions required for its adaptation to a new function, the effects of lysozyme-based and other substitutions on thermal stability were determined. Unblocking the upper cleft in alpha-lactalbumin by replacing Tyr103 with Ala, perturbs stability and structure but Pro, which also generates an open cleft, is compatible with normal structure and activity. These effects appear to reflect alternative enthalpic and entropic forms of structural stabilization by Tyr and Pro. Of 23 mutations, only three, which involve substitutions for residues in flexible substructures adjacent to the functional site, increase stability. Two are lysozyme-based substitutions for Leu110, a component of a region with alternative helix and loop conformations, and one is Asn for Lys114, a residue whose microenvironment changes when alpha-lactalbumin interacts with its target enzyme. While all substitutions for Leu110 perturb activity, a Lys114 to Asn mutation increases T(m) by more than 10 degrees C and reduces activity, but two other destabilizing substitutions do not affect activity. It is proposed that increased stability and reduced activity in Lys114Asn result from reduced flexibility in the functional site of alpha-lactalbumin.

We recently have identified an antigen receptor in sharks called NAR ( n ew or n urse shark a ntigen r eceptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, flexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (V H ) sequences prevent dimer formation with L chains. NAR also displays a uniquely flexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.

The propensity of proteins or peptides to misfold into amyloid deposits is associated with many neurodegenerative and systemic amyloid related diseases. It was originally thought that information stored in a polypeptide chain encodes only a single native fold. The transition of a protein from its soluble functional form into the β-sheet-rich insoluble ordered filamentous polymer is a special form of misfolding which poses a great intellectual challenge to the scientific community which seeks to understand the underlying mechanism. It is now believed that amyloid fibrillation may be a separate alternative folding pathway. This process is not unique to any specific class of proteins and appears to be an alternative conformation of the polypeptide backbone, in which fibrillation conditions are dictated by the amino acid sequence. Unlike native protein folding, in fibrillation, a single polypeptide chain can produce polymorphic fibrils that differ in intra- or inter-residue interactions, and which result in variations of filament number, arrangement and conformation. In this chapter we survey all the α-helical proteins that have been shown to undergo this transition to the amyloid-like form. Moreover, we discuss the unique polymorphisms that may be associated with each α-helical protein, and advances in our understanding of the transition of helical proteins into amyloid fibrils.

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.

There is no simple answer to the question ‘what is life?’ The question is itself open to all sorts of interpretations that range from the strictly biochemical to the profoundly philosophical. Although the attributes of a living organism may at one level appear intuitive, they are actually very difficult to set down with economy in such a way as to encompass all known life forms. Paradoxically, it might be those engaged in the search for extra-terrestrial life who, not being bound to what might just turn out to be terrestrial adaptations, succeed in arriving at a truly universal definition of life. Returning to Earth, we must always guard against accepting the commonly observed as being definitive: just last year a bacterium was isolated from a lake in California in which the phosphorus present in its DNA could be substituted for by arsenic (Wolfe-Simon et al., 2010). We are therefore obliged to proceed without the luxury of a crisp definition of life, but that need not impede our objectives here which are of a strictly utilitarian purpose; to provide our readers – who we assume are engineers and physical scientists – with an appreciation of the nature of living organisms. These engineers and scientists share at least one thing in common – an interest in the interaction of atmospheric gas plasmas with living organisms, and although this puts in to context our objectives, we will only have cause to mention gas plasmas at the very end of this chapter. In attempting to meet our objectives we are mindful of the limitations imposed upon us; we have but one chapter in which to convey what the essence of living organisms is.

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 β-sheet amyloid fibril, using a comprehensive range of spectroscopic techniques that provide insight into this process. This transition from α-helical to β-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 β-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.

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-β-sheet immunoglobulin and mixed α/β-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.

Amyloid fibrils are ordered beta-sheet aggregates that are associated with a number of neurodegenerative diseases such as Alzheimer's and Parkinson's. There are approximately twenty proteins that have been found to form fibrils in humans and are associated with disease states. However, it has been postulated that all proteins can be induced to form this low energy state if subjected to the right conditions. We are investigating potential methods that will lead to the destruction of fibrils, formed by the protein alpha-synuclein, which underlie Parkinson's disease. At present there is no cure for this progressive and debilitating disease. Initial studies by the Greene and Laroussi groups indicate that cold plasma can break these alpha-synuclein fibrils into smaller units in vitro. This avenue of research may facilitate the development of a medical treatment. In this study, the plasma is generated by the plasma pencil, a device capable of emitting a plasma plume several centimeters in length. The plasma pencil is powered by high voltage short pulses (nanoseconds to microseconds) and uses helium as a feed gas. The plasma plume is at room temperature but generates reactive oxygen and nitrogen species (along with charged particles) that interact with the fibrils and ultimately causing their destruction.

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.

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.

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.

Retinol-binding protein transports retinol, and circulates in the plasma as a macromolecular complex with the protein transthyretin. Under acidic conditions retinol-binding protein undergoes a transition to the molten globule state, and releases the bound retinol ligand. A biased molecular dynamics simulation method has been used to generate models for the ensemble of conformers populated within this molten globule state. Simulation conformers, with a radius of gyration at least 1.1 A greater than that of the native state, contain on average 37%beta-sheet secondary structure. In these conformers the central regions of the two orthogonal beta-sheets that make up the beta-barrel in the native protein are highly persistent. However, there are sizable fluctuations for residues in the outer regions of the beta-sheets, and large variations in side chain packing even in the protein core. Significant conformational changes are seen in the simulation conformers for residues 85-104 (beta-strands E and F and the E-F loop). These changes give an opening of the retinol-binding site. Comparisons with experimental data suggest that the unfolding in this region may provide a mechanism by which the complex of retinol-binding protein and transthyretin dissociates, and retinol is released at the cell surface.

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

Amyloid fibrils are ordered beta-sheet aggregates that are associated with a number of neurodegenerative diseases such as Alzheimer and Parkinson. At present, there is no cure for these progressive and debilitating diseases. Here we report initial studies that indicate that low temperature atmospheric pressure plasma can break amyloid fibrils into smaller units in vitro. The plasma was generated by the “plasma pencil,” a device capable of emitting a long, low temperature plasma plume/jet. This avenue of research may facilitate the development of a plasma-based medical treatment.

We systematically identify a group of evolutionarily conserved residues proposed for folding in a model β-barrel superfamily, the lipocalins. The nature of conservation at the structural level is defined and we show that the conserved residues are involved in a network of interactions that form the core of the fold. Exploratory kinetic studies are conducted with a model superfamily member, human serum retinol-binding protein, to examine their role. The present results, coupled with key experimental studies conducted with another lipocalin β-lactoglobulin, suggest that the evolutionarily conserved regions fold on a faster folding time-scale than the non-conserved regions.

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.