206 An artificial active DNA transporter from biological building blocks

The selective transport of molecules across membrane pores is an essential biological process that occurs in all living organisms. Examples include the chaperone of proteins and mRNA in and out the cell nucleus mediated by the nuclear pore complex and the transport of specific molecules across a biological membrane mediated by active and passive transporters. Here, I describe an artificial DNA transporter that is formed by incorporating a DNA carrier element into a biological nanopore imbedded in a lipid bilayer. This device is able to transport specific DNA strands across a biological membrane mediated by a simple reaction mechanism based on DNA strand displacement. Similar to biological secondary active transporters, this system uses an electrochemical potential difference to pump a specific substrate across a biological membrane at a constant transmembrane potential. Our DNA actuator might be used to separate or concentrate nucleic acids, or to vehicle genetic information across the biological membranes.     

82 Targeting intramolecular DNA structures with complementary strands

Antisense, antigene, and siRNA strategies are currently used to control the expression of genes. To this end, our laboratory is mimicking the targeting of mRNA by reacting DNA stem-loop motifs with their partially complementary strands. Specifically, we used a combination of isothermal titration (ITC), differential scanning calorimetry (DSC), and temperature-dependent UV spectroscopy to investigate: (1) the unfolding of a pseudoknot and a complex containing joined triplex-duplex motifs (shown below); and (2) the reaction of these compact structures with single strands that are complementary to the bases in the loops and to a portion of their stem.     

12 Reconstructing the RNA world

A critical event in the origin of life is thought to have been the emergence of an RNA molecule capable of self-replication as well as mutation, and hence evolution towards ever more efficient replication. As this primordial replicase appears to have been lost in time, we use synthetic biology to build modern-day “Doppelgangers” of the ancestral replicase to reconstruct and study their properties in an effort to learn more about life’s first genetic system. I will discuss our progress in the engineering and evolution of RNA polymerase ribozymes as well as the potential role that structured media such as the eutectic phase of water–ice may have played in the emergence of RNA self-replication.     

204 Polymer translocation

The translocation of a single macromolecule through a protein pore or a solid-state nanopore involves three major stages: (1) approach of the macromolecule towards the pore, (2) capture/recognition of the macromolecule at the pore entrance, and (3) threading through the pore (see the Figure) (Muthukumar, 2011). All of these stages are controlled by conformational entropy of the macromolecule, charge decoration, and the geometry of the pore, hydrodynamics, and electrostatic interactions. Chief among the contributing factors are the entropic barrier presented by the pore to the penetration of the macromolecule, pore–polymer interactions, electro-osmotic flow, and the drift-diffusion of the macromolecule in electrolyte solutions. A unifying theory of these contributing factors will be described in the context of a few illustrative experimental data on DNA translocation and protein translocation through protein pores and solid-state nanopores. Future challenges to specific biological systems will be briefly discussed.     

152 Order from disorder: defining structure in disordered proteins

Intrinsically disordered proteins are involved in a range of functional roles in the cell, as well as being associated with a number of diverse diseases, including cancers, neurodegenerative disorders, and cardiac myopathies. We use single-molecule fluorescence approaches to characterize disordered proteins implicated in the progression of Parkinson’s and Alzheimer’s diseases. Our goal is to understand, how disease-associated modifications to these proteins alter their conformational and dynamic properties and to relate these changes to disease pathology.     

210 Computational study of substrates and mediators features of lacasses

Laccases are enzymes of the family multicopper oxidases, being widely used for biotechnological applications (Canas & Camarero, 2010). The enzymes’ catalytic cycle consists of the oxidation of the substrate with the concomitant reduction of molecular oxygen to water. In this process, the substrate is converted to a free radical, that can oxidize larger substrates acting as a mediator or it can undergo polymerization. Substrate binding is not specific, and there is a large diversity of substrates for laccases. Moreover, the binding site shows important differences among diverse species. The goal of the present work is to characterize the laccase binding pocket of different species, in order to establish their common pharmacophoric characteristics. For this purpose, we have carried out docking studies with a subset of substrates, covering the diversity of substrates using the Glide program (Friesner et al., 2004). We have also analyzed the characteristics of the binding site using diverse probes. We further have rationalized the differential values of km found among diverse species for a specific substrate. Finally, special attention has been devoted to the binding of the mediator 2,2′-azido-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), commonly used in industrial processes. Figure 1 shows, ABTS docked onto the fungal laccase, whereas Figure 2 shows ABTS docked onto the bacterial laccase. The analysis of the protein–ligand complex together with the corresponding optimized geometries of the possible substrate species carried out using DFT suggest that the bound species is the protonated form of ABTS as previously suggested (Enguita et al., 2004). Furthermore, the results of this study also suggest that its mechanism of oxidation occurs in a similar way to the rest of substrates/mediators, in contrast to previous suggestions (Fabbrini et al., 2002).      

169 Polymorphic assembly of computationally designed hydrophobic-rich collagen peptides

We seek to understand how the position and length of hydrophobic content within a collagen peptide sequence dictates morphology of self-assembly. We modeled collagen assembly using diffusion limited aggregation¹ (DLA) (Parkinson et al. 1995). of discretized, rigid rods composed of hydrophilic and hydrophobic spheres. Simulations predicted that the inclusion of short hydrophobic domains should direct the assembly of lamellar structures. We designed a set of collagen peptide sequences with six, five and four contiguous nonpolar residues. Electron microscopy of aggregates revealed the peptide with six nonpolar residues self-assembled into uniform fibrils and the peptide with five residues assembled into both fibrils and plates, while including four hydrophobic residues that formed only plates. This polymorphic behavior can be explained by packing models of rod vs. screw-like-particles.     

121 Influence of changes in DNA conformation on thermodynamic contribution during interaction of human genomic DNA and its mutant with anticancer drugs

Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012).     

116 Deamination of both methyl- and normal-cytosine by the foreign DNA restriction enzyme APOBEC3A

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.     

7 Imperfect RNA synthesis via model prebiotic reactions and consequences for functional RNAs

Contemporary life synthesizes RNA of homogeneous length and regioisomer composition via sophisticated enzymatic catalysis. Before such catalysts existed, RNA could have been produced only via simpler, non-enzymatic means, which model prebiotic systems have shown produce pools of products that are similar, but varied (e.g. in regioisomer composition). Recently, we have demonstrated that functional RNAs (ribozymes and aptamers) containing mixed-regioisomer backbones (i.e. 2′–5′ vs. 3′–5′ linkages) retain function. This observation, coupled with the well-known fact that mixed-regioisomer RNAs exhibit depressed melting temperatures relative to native RNA, suggests that mixed-regioisomer backbones could actually be adaptive in an RNA (or pre-RNA) world. In this poster, we will show our recent work with functional RNAs representative of those produced in non-enzymatic polymerization reactions and their behaviours as catalysts and receptors.     

203 Polymers through pores: single-molecule experiments with nucleic acids,polypeptides and polysaccharides

The translocation of polymers through pores has been examined for almost two decades with an emphasis on nucleic acids. There are also interesting circumstances in biology where polypeptides and polysaccharides pass through transmembrane pores, and our laboratory has been investigating examples of them. Single-molecule nucleic acid sequencing by nanopore technology is an emerging approach for ultrarapid genomics. Strand sequencing with engineered protein nanopores is a viable technology which has required advances in four areas: nucleic acid threading, nucleobase identification, controlled strand translocation, and nanopore arrays (Bayley, 2012). The latter remain a pressing need and our attempts to improve arrays will be described. In several physiological situations, folded proteins pass through transmembrane pores. We have developed a model system comprising mutant thioredoxins as the translocated proteins, and staphylococcal alpha-hemolysin, as the pore. Our findings support a mechanism in which there is local unfolding near the terminus of the polypeptide that enters the pore. The remainder of the protein then unfolds spontaneously and diffuses through the pore into the recipient compartment (Rodriguez-Larrea & Bayley, 2013). We have also examined the pore formed by the E. coli outer membrane protein Wza, which transports capsular polysaccharide from its site of synthesis to the outside of the cell. We made mutant open forms of the pore and screened blockers for them by electrical recording in planar bilayers. The most effective blocker binds in the alpha-helix barrel of Wza, a site accessible from the external medium, and therefore, a prospective target for antibiotics (Kong et al., 2013).     

212 Shifting interfaces: changes in protein–protein and protein–DNA interfaces probed via molecular dynamics

Over the past decade, there has been a growing interest in studying the binding of DNA to the MutSalpha protein complex. This heterodimeric protein complex, the Msh2/Msh6 complex in humans, is the initial complex that binds mismatched DNA and other DNA defects that occur during replication. This complex has also been shown to bind at least some types of damaged DNA, such as the cross-linked adducts due to the chemotherapeutics cisplatin and carboplatin, or the incorporation of the chemotherapeutic, FdU. As a result of this interest, multiple studies have contrasted the interactions of MutSalpha with its normal mismatched substrate and with the interactions of MutsSalpha with the DNA damaged by chemotherapeutic cisplatin. To complement these studies, we examine the interaction between MutSalpha and the DNA damaged by carboplatin via all-atom molecular dynamics simulations. These simulations provide evidence for subtle changes in the protein–DNA and protein–protein interfaces. The interfaces shifts found are broadly similar to those found in binding with adduct from cis-platin, but have distinct differences. These subtle differences may play a role in the way of the different damages and mismatched DNA are signaled by MutSalpha, and suggest a signaling mechanism for DNA damage that chiefly involves shifts in protein–protein interactions as opposed to changes in protein conformation.     

101 Exploring the 3D spatial organization of the genome

Knowledge about the 3D organization of the genome will offer great insights into how cells retrieve and process the genetic information. Knowing the spatial probability distributions of individual genes will provide insights into gene regulatory and replication processes, and fill in the missing links between epigenomics, functional genomics, and structural biology. We will discuss an approach to determine 3D genome structures and structure–function maps of genomes by integrating divers types of data. To address the challenge of modeling highly variable genome structures, we discuss a population-based modeling approach, where we construct a large population of 3D genome structures that together are entirely consistent with all available experimental data including data from genome-wide chromosome conformation capture and imaging experiments. We interpret the result in terms of probabilities of a sample drawn from a population of heterogeneous structures. We will discuss results on the 3D spatial organization of genomes in human lymphoblastoid cells and budding yeast.     

123 Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology

Deregulation of mini-chromosome maintenance (MCM) proteins are associated with genomic instability and cellular abnormality. MCM complexes are recruited to replication origins for S phase genome duplication. Paradoxically, MCM proteins are expressed in large number of origins and are associated with unreplicated chromatin regions away from the origins during G1 and S phases. We report an unusually wide left-handed filament structure for an archeal MCM, as determined by X-ray and electron microscopy. The crystal structure reveals that an α-helix bundle formed between two neighboring subunits plays a critical role in filament formation which we show through mutation and electron microscopy. The filament interior has a remarkably strong electro-positive surface spiraling along the inner filament channel which we establish as the binding site for double-stranded DNA. We find that MCM filament binding to DNA causes dramatic topological changes which negatively supercoil circular DNA through inducing loosening of the double helix. This newly identified biochemical activity of MCM may imply a wider functional role for MCM in DNA metabolism beyond helicase function, for the non-origin bound MCMs. Finally, using yeast genetics, we show that the inter–subunit interactions important for MCM filament formation play a role for cell growth and survival.     

187 Plucking the high hanging fruit: a systematic approach for targeting protein interfaces

Development of specific ligands for protein targets that help decode the complexities of protein–protein interaction networks is a key goal for the field of chemical biology. Despite the emergence of powerful in silico and experimental high-throughput screening strategies, the discovery of synthetic ligands that selectively modulate protein–protein interactions remains a challenge for the chemical biologists. Proteins often utilize small folded domains for recognition of other biomolecules. The basic hypothesis guiding our research is that by mimicking these domains, we can modulate the function of a particular protein with metabolically-stable synthetic molecules (Raj et al., 2013). This presentation will discuss computational approaches (Bullock et al., 2011; Jochim & Arora, 2010) to identify targetable interfaces along with synthetic methods (Patgiri et al., 2008; Tosovska & Arora, 2010) to develop protein domain mimics (PDMs) as modulators of intracellular protein–protein interactions (Henchey et al., 2010; Patgiri et al., 2011).     

164 Extracting dynamics information from multiple structures

Meaningful dynamics information can be extracted from multiple experimental structures of the same, or closely related, proteins or RNAs. The covariance matrix of atom positions is decomposable into its principal components, and in this way, it is possible to rank-order the changes in the set of structures, and to determine what the most significant changes are. Usually, only a few principal components dominate the motions of the structures, and these usually relate to the functional dynamics. This dynamics information provides strong evidence for the plasticity of protein and RNA structures, and also suggests that these structures almost always have a highly limited repertoire of motions. In some cases, such as HIV protease, the dominant motions are opening and closing over the active site. For myoglobin, the changes are much smaller, reflecting in part the small changes in sequence, but nonetheless they show characteristic details that depend on the species. Sets of structures can also be used to derive the effective microscopic forces that are forcing a given conformational transition.     

66 Architectural role of HMO1 in bending,bridging, and compacting DNA

HMO1 proteins are abundant Saccharomyces cerevisiae (yeast) High Mobility Group Box (HMGB) protein (Kamau, Bauerla & Grove, 2004). HMGB proteins are nuclear proteins which are known to be architectural proteins (Travers, 2003). HMO1 possesses two HMGB box domains. It has been reported that double box HMGB proteins induce strong bends upon binding to DNA. It is also believed that they play an essential role in reorganizing chromatin and, therefore, are likely to be involved in gene activation. To characterize DNA binding we combine single molecule stretching experiments and AFM imaging of HMO1 proteins bound to DNA. By stretching DNA bound to HMO1, we determine the dissociation constant, measure protein induced average DNA bending angles, and determine the rate at which torsional constraint of the DNA is released by the protein. To further investigate the local nature of the binding, AFM images of HMO1-DNA complexes are imaged, and we probe the behavior of these complexes as a function of protein concentration. The results show that at lower concentrations, HMO1 preferentially binds to the ends of the double helix and links to the separate DNA strands. At higher concentrations HMO1 induces formation of a complex network that reorganizes DNA. Although HMG nuclear proteins are under intense investigation, little is known about HMO1. Our studies suggest that HMO1 proteins may facilitate interactions between multiple DNA molecules.     

102 Genome engineering nucleases derived from GIY-YIG homing endonucleases

Efficient targeted manipulation of complex genomes requires highly specific endonucleases to generate double-strand breaks at defined locations (Bibikova et al., 2003; Bogdanove and Voytas, 2011). The predominantly engineered nucleases, zinc-finger nucleases (ZFNs), and TAL effector nucleases (TALENs) use the catalytic domain of FokI as the nuclease portion. This domain, however, functions as a dimer to nonspecifically cleave DNA meaning that ZFNs and TALENs must be designed in head-to-head pairs to target a desired sequence. To overcome this limitation and expand the toolbox of genome editing reagents, we used the N-terminal catalytic domain and interdomain linker of the monomeric GIY-YIG homing endonuclease I-TevI to create I-TevI-zinc-fingers (Tev-ZFEs), and I-TevI-TAL effectors (Tev-TALs) (Kleinstiver et al. 2012). We also made I-TevI fusions to LAGLIDADGs homing endonucleases (I-Tev-LHEs). All the three fusions showed activity on model substrates on par with ZFNs and TALENs in yeast-based recombination assays. These proof-of-concept experiments demonstrate that the catalytic domain of GIY-YIG homing endonucleases can be targeted to relevant loci by fusing the domain to characterize DNA-binding platforms. Recent efforts have focused on improving the Tev-TAL platform by (1) understanding the spacing requirements between the nuclease cleavage site and the DNA binding site, (2) probing the DNA binding requirements of the I-TevI linker domain, and (3) demonstrating activity in mammalian systems.