The crystal structure of restriction endonuclease Bam HI complexed to DNA has been determined at 2.2 angstrom resolution. The DNA binds in the cleft and retains a B-DNA type of conformation. The ...enzyme, however, undergoes a series of conformational changes, including rotation of subunits and folding of disordered regions. The most striking conformational change is the unraveling of carboxyl-terminal α helices to form partially disordered "arms." The arm from one subunit fits into the minor groove while the arm from the symmetry related subunit follows the DNA sugar-phosphate backbone. Recognition of DNA base pairs occurs primarily in the major groove, with a few interactions occurring in the minor groove. Tightly bound water molecules play an equally important role as side chain and main chain atoms in the recognition of base pairs. The complex also provides new insights into the mechanism by which the enzyme catalyzes the hydrolysis of DNA phosphodiester groups.
FokI is a member of an unusual class of bipartite restriction enzymes that recognize a specific DNA sequence and cleave DNA nonspecifically a short distance away from that sequence. Because of its ...unusual bipartite nature, FokI has been used to create artificial enzymes with new specificities. We have determined the crystal structure at 2.8A resolution of the complete FokI enzyme bound to DNA. As anticipated, the enzyme contains amino- and carboxy-terminal domains corresponding to the DNA-recognition and cleavage functions, respectively. The recognition domain is made of three smaller subdomains (D1, D2 and D3) which are evolutionarily related to the helix-turn-helix-containing DNA-binding domain of the catabolite gene activator protein CAP. The CAP core has been extensively embellished in the first two subdomains, whereas in the third subdomain it has been co-opted for protein-protein interactions. Surprisingly, the cleavage domain contains only a single catalytic centre, raising the question of how monomeric FokI manages to cleave both DNA strands. Unexpectedly, the cleavage domain is sequestered in a 'piggyback' fashion by the recognition domain. The structure suggests a new mechanism for nuclease activation and provides a framework for the design of chimaeric enzymes with altered specificities.
Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and ...no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.
The crystal structure of the HincII restriction endonuclease-DNA complex shows that degenerate specificity for blunt-ended cleavage at GTPyPuAC sequences arises from indirect readout of ...conformational preferences at the center pyrimidine-purine step. Protein-induced distortion of the DNA is accomplished by intercalation of glutamine side chains into the major groove on either side of the recognition site, generating bending by either tilt or roll at three distinct loci. The intercalated side chains propagate a concerted shift of all six target-site base pairs toward the minor groove, producing an unusual cross-strand purine stacking at the center pyrimidine-purine step. Comparison of the HincII and EcoRV cocrystal structures suggests that sequence-dependent differences in base-stacking free energies are a crucial underlying factor mediating protein recognition by indirect readout.
The interaction of BamHI endonuclease with DNA has been studied crystallographically, but has not been characterized rigorously in solution. The enzyme binds in solution as a homodimer to its ...recognition site GGATCC. Only six base-pairs are directly recognized, but binding affinity (in the absence of the catalytic cofactor Mg2+) increases 5400-fold as oligonucleotide length increases from 10 to 14 bp. Binding is modulated by sequence context outside the recognition site, varying about 30-fold from the bes t (GTG or TAT) to the worst (CGG) flanking triplets.BamHI,EcoRI and EcoRV endonucleases all have different context preferences, suggesting that context affects binding by influencing the free energy levels of the complexes rather than that of the free DNA. Ethylation interference footprinting in the absence of divalent metal shows a localized and symmetrical pattern of phosphate contacts, with strong contacts at NpNpNpGGApTCC. In the presence of Mg2+, first-order cleavage rate constants are identical in the two GGA half-sites, are the same for the two nicked intermediates and are unaffected by substrate length in the range 10-24 bp. DNA binding is strongly enhanced by mutations D94N, E111A or E113K, by binding of Ca2+ at the active site, or by deletion of the scissile phosphate GpGATCC, indicating that a cluster of negative charges at the catalytic site contributes at least 3-4 kcal/mol of unfavorable binding free energy. This electrostatic repulsion destabilizes the enzyme-DNA complex and favors metal ion binding and progression to the transition state for cleavage.
The μ-opioid receptor mediates not only the beneficial painkilling effects of opiates like morphine but also the detrimental effects of chronic exposure such as tolerance and dependence. Different ...studies have linked tolerance to opioid receptor desensitization. Agonist activation of the μ-opioid receptor stimulates a mitogen-activated protein kinase (MAPK) activity, but the functional significance of this pathway remains unclear. We have focused on the MAPK signaling cascade to study μ-opioid receptor desensitization. We report that inhibition of the MAPK pathway blocks desensitization of μ-opioid receptor signaling as well as the loss of receptor density due to internalization. Our results suggest that a feedback signal emanating from the MAPK cascade is required for μ-opioid receptor desensitization.
Interfacial supramolecular electrochemistry Cui, Kang; Dorner, Iris; Mertens, Stijn F.L.
Current opinion in electrochemistry,
March 2018, 2018-03-00, Letnik:
8
Journal Article
Recenzirano
•Key principles of self-assembly at electrified solid–liquid interfaces are reviewed.•The effect of the electric field in the double layer on intermolecular and substrate–molecule interactions is ...explained.•The equivalence between the electrochemical interface and the field in an STM configuration is analyzed.
Supramolecular chemistry at solid–liquid interfaces is guided by the interactions between the molecular building blocks, the solid substrate and the liquid phase. At an electrochemical interface (i.e., at the interface between an electronic and ionic conductor), the substrate potential allows modulating many of these interactions, resulting in a high level of control over the supramolecular structures that are formed and their reactivity. In this paper, we review key principles and recent work in this area, and discuss how a standard scanning tunneling microscope setup allows to scale down interfacial supramolecular electrochemistry to the few-molecule level.
Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was ...demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antlsense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E.coil carrying the aadA gene construct, and trartsformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
Here, we investigate the highly specific interaction of the BamHI endonuclease with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA ...interface while maintaining specificity. Mutational studies, together with the structural determination of the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the specific binding of the enzyme to its cognate DNA recognition sequence. Amino acid residues N116, S118, R122, D154 and R155 are involved in DNA sequence recognition and are located in the major groove in close proximity to the nucleotide bases comprising the recognition sequence. Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference selection, was used to identify an array of substitutions which maintain site-specific binding to the cognate GGATCC sequence. This approach has demonstrated the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding. One variant, double mutant N116H, S118G was found to cleave DNA only when the adenine base in the recognition site is methylated.
A neutrino with energy ∼290 TeV, IceCube-170922A, was detected in coincidence with the BL Lac object TXS 0506+056 during enhanced gamma-ray activity, with chance coincidence being rejected at ∼3 ...level. We monitored the object in the very-high-energy (VHE) band with the Major Atmospheric Gamma-ray Imaging Cherenkov (MAGIC) telescopes for ∼41 hr from 1.3 to 40.4 days after the neutrino detection. Day-timescale variability is clearly resolved. We interpret the quasi-simultaneous neutrino and broadband electromagnetic observations with a novel one-zone lepto-hadronic model, based on interactions of electrons and protons co-accelerated in the jet with external photons originating from a slow-moving plasma sheath surrounding the faster jet spine. We can reproduce the multiwavelength spectra of TXS 0506+056 with neutrino rate and energy compatible with IceCube-170922A, and with plausible values for the jet power of . The steep spectrum observed by MAGIC is concordant with internal γγ absorption above ∼100 GeV entailed by photohadronic production of a ∼290 TeV neutrino, corroborating a genuine connection between the multi-messenger signals. In contrast to previous predictions of predominantly hadronic emission from neutrino sources, the gamma-rays can be mostly ascribed to inverse Compton upscattering of external photons by accelerated electrons. The X-ray and VHE bands provide crucial constraints on the emission from both accelerated electrons and protons. We infer that the maximum energy of protons in the jet comoving frame can be in the range ∼1014 - 1018 eV.
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