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TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.

The crystal structure of the AvrBs3–DNA complex is reported., Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein–DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152–895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold.

We report on several proteins recently solved by structural genomics consortia, in particular by the Northeast Structural Genomics consortium (NESG). The proteins considered in this study differ substantially in their sequences but they share a similar structural core, characterized by a pseudobarrel five-stranded beta sheet. This core corresponds to the PUA domain-like architecture in the SCOP database. By connecting sequence information with structural knowledge, we characterize a new subgroup of these proteins that we propose to be distinctly different from previously described PUA domain-like domains such as PUA proper or ASCH. We refer to these newly defined domains as EVE. Although EVE may have retained the ability of PUA domains to bind RNA, the available experimental and computational data suggests that both the details of its molecular function and its cellular function differ from those of other PUA domain-like domains. This study of EVE and its relatives illustrates how the combination of structure and genomics creates new insights by connecting a cornucopia of structures that map to the same evolutionary potential. Primary sequence information alone would have not been sufficient to reveal these evolutionary links.

Malaria is caused by protozoan parasites of the genus Plasmodium. Four species of Plasmodium can infect humans: P. falciparum, P. malariae, P. vivax, and P. ovale. P. falciparum is the only able to cytoadhere to the surface of postcapillary endothelial cells. A key role in cytoadherence is played by the interaction between the PfEMP1 P. falciparum protein and the human intracellular adhesion molecule (ICAM-1) although very little is known about the molecular details of this complex. Here we propose a model for this interaction on the basis of a homology model of the functional domain of PfEMP1 and of the ICAM-1 three dimensional structures. Our model is consistent with the results of many experimental observations, provides a rational explanation for the different binding abilities of different strains of P. falciparum and explains the reduced binding affinity of the A4 strain of P. falciparum for the ICAM-1Kilifi polymorphism. On the basis of our model, we can also explain why the murine ICAM-1, although sharing 70% sequence similarity with its human homologue, does not bind PfEMP1, and why the binding of fibrinogen and PfEMP1 to ICAM-1 is mutually exclusive. The model of the complex proposed here can serve as a useful tool for the design and interpretation of biochemical and immunological experimental results. Proteins 2007. © 2007 Wiley-Liss, Inc.

The effect of increasing concentrations of several anions on the azide (N − 3 ) binding properties of sperm whale and horse ferric myoglobin has been studied. Surprisingly, a number of anions may act as heterotropic effectors, decreasing the affinity of myoglobins for N − 3 , in the following order: ClO − 4 =I − >Br − >Cl − and SO 2− 4 , which mirrors the increase in their charge density. The largest effects were measured using ClO − 4 and I − , which produce a 4-fold and 8-fold reduction of the N − 3 binding affinity in horse and sperm whale myoglobins, respectively. A dissociation equilibrium constant ( K d ) ranging from 150 to 250 mM was estimated for ClO − 4 and I − binding to myoglobins. In order to analyse the molecular mechanism producing the reduction of the N − 3 binding affinity to ferric myoglobin, the potential anionic binding sites within ferric myoglobin were investigated by a molecular modelling study using the program Grid. Analysis of the theoretical results suggests two particularly favourable binding sites: the first, next to the distal side of the haem, whose occupancy might alter the electrostatic potential surrounding the bound N − 3 ; the second, involving residues of helices B and G which are far from the haem iron atom, thus implying a long range effect on the bound N − 3 . Based on the evidence that no significant conformational changes are found in the three-dimensional structures of N − 3 -free and N − 3 -bound myoglobin and on previous results on N − 3 binding to ferric myoglobin mutants in CD3 positions, we favour the first hypothesis, suggesting that the functional heterotropic modulation of monomeric myoglobin is mainly depending on a decrease of the positive charge density induced by the binding of anions to the haem distal side.

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator-like effector nucleases (TALEN). The analysis of > 15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only accommodate a relatively small number of position-dependent mismatches while maintaining a detectable activity at endogenous loci in vivo, demonstrating the high specificity of these molecular tools. We thus envision that the results we provide will allow for more deliberate choices of DNA binding arrays and/or DNA targets, extending our engineering capabilities.

The last few years have seen the increasing development of new DNA targeting molecular tools and strategies for precise genome editing. However, opportunities subsist to either improve or expand the current toolbox and further broaden the scope of possible biotechnological applications. Here we report the discovery and the characterization of BurrH, a new modular DNA binding protein from Burkholderia rhizoxinica that is composed of highly polymorphic DNA targeting modules. We also engineered this scaffold to create a new class of designer nucleases that can be used to efficiently induce in vivo targeted mutagenesis and targeted gene insertion at a desired locus.

Background: Meganucleases are important tools for genome engineering, providing an efficient way to generate DNA double-strand breaks at specific loci of interest. Numerous experimental efforts, ranging from in vivo selection to in silico modeling, have been made to re-engineer meganucleases to target relevant DNA sequences. Results: Here we present a novel in silico method for designing custom meganucleases that is based on the use of a machine learning approach. We compared it with existing in silico physical models and high-throughput experimental screening. The machine learning model was used to successfully predict active meganucleases for 53 new DNA targets. Conclusions: This new method shows competitive performance compared with state-of-the-art in silico physical models, with up to a fourfold increase in terms of the design success rate. Compared to experimental high-throughput screening methods, it reduces the number of screening experiments needed by a factor of more than 100 without affecting final performance.

Methods and kits for detecting nucleic acid sequences of interest using dna-binding protein domain

Crystal structures of BurrH and the BurrH–DNA complex are reported., DNA editing offers new possibilities in synthetic biology and biomedicine for modulation or modification of cellular functions to organisms. However, inaccuracy in this process may lead to genome damage. To address this important problem, a strategy allowing specific gene modification has been achieved through the addition, removal or exchange of DNA sequences using customized proteins and the endogenous DNA-repair machinery. Therefore, the engineering of specific protein–DNA interactions in protein scaffolds is key to providing ‘toolkits’ for precise genome modification or regulation of gene expression. In a search for putative DNA-binding domains, BurrH, a protein that recognizes a 19 bp DNA target, was identified. Here, its apo and DNA-bound crystal structures are reported, revealing a central region containing 19 repeats of a helix–loop–helix modular domain (BurrH domain; BuD), which identifies the DNA target by a single residue-to-nucleotide code, thus facilitating its redesign for gene targeting. New DNA-binding specificities have been engineered in this template, showing that BuD-derived nucleases (BuDNs) induce high levels of gene targeting in a locus of the human haemoglobin β (HBB) gene close to mutations responsible for sickle-cell anaemia. Hence, the unique combination of high efficiency and specificity of the BuD arrays can push forward diverse genome-modification approaches for cell or organism redesign, opening new avenues for gene editing.

The structural and oxygen binding properties of Acipenser naccarii blood have been investigated. The electrophoretic analysis of the hemolysate of this sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. Constituent globin chains were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography. N-terminal amino acid sequence analysis revealed the presence of a N-terminal proline in two of the three α-chains present in the globin pattern, and the presence of a histidine residue in 2βposition. Oxygen equilibria reveal a very low sensitivity of the individual hemoglobins to chloride ions and temperature; however, in the presence of organic phosphates the oxygen affinity of the hemoglobin components decreases strongly. In particular, when Guanosine-5-triphosphate (GTP) is added, the reduction of the oxygen affinity, at pH 7.4 and 20 °C, is 60% and 50%, respectively, for HbI (anodic component) and HbII (cathodic component). As the effect of protons is concerned, the small Root effect shown by total hemolysate at physiological conditions, seems to be due mainly to the cathodic component. On the whole, the functional properties shown by sturgeon hemoglobin components seem to be related to the particular physiological needs dictated by the environmental characteristics.

International audience; Netherton syndrome is caused by loss-of-function mutations in SPINK5 encoding the Kazal-type inhibitor LEKTI-1 leading to dysregulation of proteolytic cascades involving several kallikreins. We used both structure-based and ligand-based virtual screening computations to identify commercially available non-covalent inhibitors of human kallikrein 5 (hK5), a serine protease (trypsin-like) that plays a central role in the initiation of the molecular cascades leading to the Netherton syndrome phenotype. The efficacy and mechanism of inhibition of the identified new families of organic compounds were analyzed not only for hK5 but also on other proteases implicated in the cascades (hK7, hK14 and matriptase). These inhibitors are nontoxic on healthy human keratinocytes and are structurally different from traditional serine protease inhibitors validating their potential utility as initial hits to control proteolytic disorders observed in dermatological pathologies such as Netherton syndrome.

The thermal response of three proteins with mesophilic, thermophilic and hyperthermophilic character hints at the essential role played in thermostability by the protein-water interface. The formation of spanning water clusters enveloping the macromolecule and their resistance to thermal stress is shown to correlate with the charge distribution at the protein surface; in particular our findings suggest an effective role of the superficial charge distribution in stabilizing the global connectivity of the hydration water.

Three homologous proteins with mesophilic, thermophilic and hyperthermophilic character have been studied via molecular dynamics simulations at four different temperatures in order to investigate how water controls thermostability. The water-exposed surface of the protein is shown to increase with the degree of thermophilicity, and the role of water in enhancing the protein internal flexibility and structural robustness is elucidated. The presence of water-water hydrogen bond clusters enveloping the macromolecule is shown to correlate with thermal robustness when going from the mesophilic to the hyperthermophilic variants. Our analysis indicates that essential contributions to thermostability stem from protein-water surface effects whereas the protein internal packing plays a minor role.

The salt dependence of the binding free energy of five protein-protein hetero-dimers and two homo-dimers/tetramers was calculated from numerical solutions to the Poisson-Boltzmann equation. Overall, the agreement with experimental values is very good. In all cases except one involving the highly charged lactoglobulin homo-dimer, increasing the salt concentration is found both experimentally and theoretically to decrease the binding affinity. To clarify the source of salt effects, the salt-dependent free energy of binding is partitioned into screening terms and to self-energy terms that involve the interaction of the charge distribution of a monomer with its own ion atmosphere. In six of the seven complexes studied, screening makes the largest contribution but self-energy effects can also be significant. The calculated salt effects are found to be insensitive to force-field parameters and to the internal dielectric constant assigned to the monomers. Nonlinearities due to high charge densities, which are extremely important in the binding of proteins to negatively charged membrane surfaces and to nucleic acids, make much smaller contributions to the protein-protein complexes studied here, with the exception of highly charged lactoglobulin dimers. Our results indicate that the Poisson-Boltzmann equation captures much of the physical basis of the nonspecific salt dependence of protein-protein complexation.

In this study a decreased DPG response by polar bear (Ursus maritimus) hemoglobin was observed, and this response was interpreted as an example of gradual DPG/chloride shifting. This sort of mechanism has been suggested to occur in ruminants and, intuitively, one might guess that for ruminants the DPG/Cl - shifting might have been beneficial and hence selected for at the time of the latest Ice Age. However, sugges- tion that this is purely a temperature effect in polar bears and ruminants conflicts with the existence, in the hot savanna, of mammals that have Hb modulated by chloride. However, acidosis effects caused by routine periods of food shortage, induced in extreme environments may explain the responses of the hemoglobins of animals adapted to extreme habitats. The chloride effect is bound to specific amino acid substi- tutions in key positions. In polar bear Hb, they are specific, additional (with respect to human HbA) O2-linked chloride binding sites located between Lys-76 (b) and Lys-8 (b). The amino acids operate as an additional H + binding site for a chloride anion. Additionally, with respect to human adult HbA, the primary structure of polar bear Hb was characterized by two substitutions in b chains: Pro-5 (A2)→ Gly and Ala-76 (E20)→ Lys. The increased flexibility of the A helix causes the lower DPG effect. We further hypothesize that the resulting widening of the central cavity allows the Lys-82 (b) terminus to be free and constitute an additional, chloride-binding site. © 2004 Elsevier SAS. All rights reserved.

Haemoglobins from mammals of sub-Arctic and Arctic species, as well as fetal human Hb, are all characterized by a significantly lower ΔH of oxygenation compared with the majority of mammalian haemoglobins from temperate species (exceptions are represented by some cold-resistant species, such as cow, horse and pig). This has been interpreted as an adaptive mechanism of great importance from a physiological point of view. To date, the molecular basis of this thermodynamic characteristic is still not known. In the present study, we show that binding of extra chloride (with respect to adult human Hb) ions to Hb would significantly contribute to lowering the overall heat of oxygenation, thus providing a molecular basis for the low effect of temperature on the oxygenation–deoxygenation cycle. To this aim, the oxygen binding properties of bovine Hb, bear (Ursus arctos) Hb and horse Hb, which are representative of this series of haemoglobins, have been studied with special regard to the effect of heterotropic ligands, such as organic phosphates (namely 2,3-diphosphoglycerate) and chloride. Functional results are consistent with a mechanism for ligand binding that involves an additional binding site for chloride ion. Analysis of computational chemistry results, obtained by the GRID program, further confirm the hypothesis that the reason for the lower ΔH of oxygenation is mainly due to an increase in the number of the oxygen-linked chloride-binding sites.

Polar bear (Ursus maritimus) hemoglobin (Hb) shows a low response to 2,3-diphosphoglycerate (2,3-DPG), compared to human Hb A0, even though these proteins have the same 2,3-DPG-binding site. In addition, polar bear Hb shows a high response to chloride and an alkaline Bohr effect (deltalog P50/deltapH) that is significantly greater than that of human Hb A0. The difference in sequence Pro (Hb A0)-->Gly (polar bear Hb) at position A2 in the A helix seems to be critical for reduced binding of 2,3-DPG. Our results also show that the A2 position may influence not only the flexibility of the A helix, but that differences in flexibility of the first turn of the A helix may affect the unloading of oxygen for the intrinsic ligand affinities of the alpha and beta chains. However, preferential binding to either chain can only take place if there is appreciable asymmetric binding of the phosphoric effector. Regarding this point, 31P NMR data suggest a loss of symmetry of the 2,3-DPG-binding site in the deoxyHb-2,3-DPG complex.

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion-exchange chromatography, were studied with special regard to the effect of chloride, 2,3-diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3-diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr--Ser) and A2 (Pro--Gly) substitutions observed in the beta chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3-diphosphoglycerate as compared with human Hb.

The structural and oxygen binding properties of Acipenser naccarii blood have been investigated. The electrophoretic analysis of the hemolysate of this sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. Constituent globin chains were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography. N-terminal amino acid sequence analysis revealed the presence of a N-terminal proline in two of the three alpha-chains present in the globin pattern, and the presence of a histidine residue in 2betaposition. Oxygen equilibria reveal a very low sensitivity of the individual hemoglobins to chloride ions and temperature; however, in the presence of organic phosphates the oxygen affinity of the hemoglobin components decreases strongly. In particular, when Guanosine-5-triphosphate (GTP) is added, the reduction of the oxygen affinity, at pH 7.4 and 20 oC, is 60% and 50%, respectively, for HbI (anodic component) and HbII (cathodic component). As the effect of protons is concerned, the small Root effect shown by total hemolysate at physiological conditions, seems to be due mainly to the cathodic component. On the whole, the functional properties shown by sturgeon hemoglobin components seem to be related to the particular physiological needs dictated by the environmental characteristics.

This study was undertaken to test the symmetry of 2,3-diphosphoglycerate (2,3-DPG) binding site in hemoglobin (Hb). From Arnone's study [A. Arnone, Nature (London) 237 (1972) 146] the 2,3-DPG binding site is located at the top of the cavity, that runs through the center of the deoxy-Hb molecule. However, it is possible that this symmetry reported by Arnone, for crystals of 2,3-DPG-Hb complex, might not be conserved in solution. In this paper, we report the 31P nuclear magnetic resonances of the 2,3-DPG interaction with Hb. The 2,3-DPG chemical shifts of the P2 and P3 resonance are both pH- and hemoglobin-dependent [protein from man, polar bear (Ursus maritimus), Arctic fox (Alopex lagopus) and bovine]. 2,3-DPG binds tightly to deoxyhemoglobin and weakly, nevertheless significantly, to oxyhemoglobin. In particular, our results suggest similar spatial position of the binding site of 2,3-DPG in both forms of Hb in solutions. However, the most unexpected result was the apparent loss of symmetry in the binding site, which might correlate with the ability of the hemoglobin to modulate its functional behavior. The different interactions of the phosphate groups indicate small differences in the quaternary structure of the different deoxy forms of hemoglobin. Given the above structural perturbation an asymmetric binding in the complex could justify, at least in part, different physiological properties of Hb. Regardless, functionally relevant effects of 2,3-DPG seem to be measured and best elucidated through solution studies.

In this study the authors attempt to correlate kinetic constants for carbamylation of AChE, by a series of carbamate inhibitors, with the conformational positioning of Trp84 in transition state complexes of the same carbamates with Torpedo AChE, as obtained by computerized molecular modelling. They present evidence for changes in the distance of the carbamates from the center of the indole ring which can be correlated with the bimolecular rate constants for inhibition. As a result the greater the distance from Trp84, the smaller the bimolecular inhibition constant value, ki (= k2/Ka), becomes. In conclusion, the value of the bimolecular rate constant for selected AChE inhibitors (structural changes that have been hypothesised or natural alkaloids of unknown activity) which possess similar size and rigidity, can be obtained. Under these conditions energy minimization alone seems to be sufficient even to accurately predict protein-substrate interactions that actually occur. Modelling studies also suggest that conformational re-orientation of Trp84 in the transition state could produce an overall movement of the Cys67-Cys94 loop.