Spectroscopic characterization of the copper(I)-thiolate cluster in the DNA-binding domain of yeast ACE1 transcription factor

AbstractA polypeptide containing the amino-terminal region of ACEI (residues 1–122; 122*), the activator of yeast Cu-methallothionein gene transcription, shows charge-transfer and metal-centered UV absorption bands, and orange luminescence which are characteristic of Cu-cysteinyl thiolate cluster structures. These spectral features are abolished by the Cu(1) complexing agents CN* and diethyldithiocarbamate or exposure to acid, but not by the Cu(II)chelator. EDTA. Binding of the polypeptide to its specific DNA recognition site, but not to calf-thymus double-stranded DNA, induces quenching of its Tyr and Cu-S cluster luminescence emission. The CD spectrum is characteristic of a tightly folded structure that may be organized around the Cu cluster

DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier

Crotamine, a 42-residue polypeptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, has been shown to be a cell-penetrating protein that targets chromosomes, carries plasmid DNA into cells, and shows specificity for actively proliferating cells. Given this potential role as a nucleic acid-delivery vector, we have studied in detail the binding of crotamine to single- and double-stranded DNAs of different lengths and base compositions over a range of ionic conditions. Agarose gel electrophoresis and ultraviolet spectrophotometry analysis indicate that complexes of crotamine with long-chain DNAs readily aggregate and precipitate at low ionic strength. This aggregation, which may be important for cellular uptake of DNA, becomes less likely with shorter chain length. 25-mer oligonucleotides do not show any evidence of such aggregation, permitting the determination of affinities and size via fluorescence quenching experiments. The polypeptide binds non-cooperatively to DNA, covering about 5 nucleotide residues when it binds to single (ss) or (ds) double stranded molecules. The affinities of the protein for ss-vs. ds-DNA are comparable, and inversely proportional to salt levels. Analysis of the dependence of affinity on [NaCl] indicates that there are a maximum of,3 ionic interactions between the protein and DNA, with some of the binding affinity attributable to non-ionic interactions. Inspection of the three-dimensional structure of the protein suggests that residues 31 to 35, Arg-Trp-Arg-Trp-Lys, could serve as a potential DNA-binding site. A hexapeptide containing this sequence displayed a lower DNA binding affinity and salt dependence as compared to the full-length protein, likely indicative of a more suitable 3D structure and the presence of accessory binding sites in the native crotamine. Taken together, the data presented here describing crotamine-DNA interactions may lend support to the design of more effective nucleic acid drug delivery vehicles which take advantage of crotamine as a carrier with specificity for actively proliferating cells. Citation: Chen P-C, Hayashi MAF, Oliveira EB, Karpel RL (2012) DNA-Interactive Properties of Crotamine, a Cell-Penetrating Polypeptide and a Potential Drug Carrier. PLoS ONE 7(11): e48913. doi:10.1371/journal.pone.0048913University of Maryland Baltimore County Designated Research Initiative Fund, an Undergraduate Research AwardUniversity of Maryland Baltimore County Designated Research Initiative Fund, an Undergraduate Research AwardFundao de Amparo a Pesquisa do Estado de So Paulo [FAPESP]Fundao de Amparo a Pesquisa do Estado de So PauloNational Council of Technological and Scientific Development [CNPq]National Council of Technological and Scientific Developmen

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the beta-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 mu g/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 mu g/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 mu g/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 mu g/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. the peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications. (C) 2012 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)University of Maryland Baltimore County Designated Research Initiative FundUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Med, BR-04044020 São Paulo, BrazilUniv São Paulo, Dept Bioquim & Imunol, BR-14049 Ribeirao Preto, BrazilUniv Maryland, Sch Med, Inst Human Virol, Baltimore, MD 21201 USAUniv Maryland, Dept Biochem & Mol Biol, Baltimore, MD 21201 USAUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, BR-04044020 São Paulo, BrazilInst Butantan, Lab Bioquim & Biofis, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ginecol, BR-04044020 São Paulo, BrazilCBA, Lab Bioquim & Biol Mol, Manaus, Amazonas, BrazilUniv Maryland Baltimore Cty, Dept Chem & Biochem, Baltimore, MD 21250 USAInst Butantan, Ctr Toxinol Aplicada CAT CEPID, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Farmacol, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Med, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Bioquim, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo UNIFESP, Dept Ginecol, BR-04044020 São Paulo, BrazilWeb of Scienc

Monitoring Cooperative Binding Using Electrochemical DNA-Based Sensors

Electrochemical DNA-based (E-DNA) sensors are utilized to detect a variety of targets including complementary DNA, small molecules, and proteins. These sensors typically employ surface-bound single-stranded oligonucleotides that are modified with a redox-active molecule on the distal 3′ terminus. Target-induced flexibility changes of the DNA probe alter the efficiency of electron transfer between the redox active methylene blue and the electrode surface, allowing for quantitative detection of target concentration. While numerous studies have utilized the specific and sensitive abilities of E-DNA sensors to quantify target concentration, no studies to date have demonstrated the ability of this class of collision-based sensors to elucidate biochemical-binding mechanisms such as cooperativity. In this study, we demonstrate that E-DNA sensors fabricated with various lengths of surface-bound oligodeoxy­thymidylate [(dT)_<i>n</i>] sensing probes are able to quantitatively distinguish between cooperative and noncooperative binding of a single-stranded DNA-binding protein. Specifically, we demonstrate that oligo­(dT) E-DNA sensors are able to quantitatively detect nM levels (50 nM–4 μM) of gene 32 protein (g32p). Furthermore, the sensors exhibit signal that is able to distinguish between the cooperative binding of the full-length g32p and the noncooperative binding of the core domain (*III) fragment to single-stranded DNA. Finally, we demonstrate that this binding is both probe-length- and ionic-strength-dependent. This study illustrates a new quantitative property of this powerful class of biosensor and represents a rapid and simple methodology for understanding protein–DNA binding mechanisms

ECT as Used in Psychiatry Temporarily Opens the Blood-Brain Barrier: Could This be Used to Better Deliver Chemotherapy for Glioblastoma

Glioblastoma remains a poor‐prognosis cancer. We review research showing evanescent opening of the blood‐brain barrier, BBB, after electroconvulsive treatment, ECT. ECT as currently used in psychiatry for treatment‐resistant depression has been in continuous use throughout the world since introduction in the late 1930’s. Post-ictal BBB opening phenomenon might be safe enough to use to deliver chemotherapeutic agents that would not otherwise cross the BBB. Although the main mass of tumor in glioblastoma has a relatively leaky BBB, the invasive paucicellular migratory microsatellite glioblastoma cells that become the origin of recurrent tumor are supplied by normal poorly‐permeable capillaries, preventing ready access of potentially useful chemotherapy drugs like doxorubicin or methotrexate. These microsatellites go on to kill. Modern ECT uses deep neuromuscular blockade and cardiovascular stabilizing drugs such that muscular contractions and increases in intracranial pressure are minimized, yet the electroencephalogram shows a typical grand mal seizure. Post‐ECT BBB opening allows transgression of > 4 kDa peptides, potentially comfortable enough to give free access to brain tissue of doxorubicin or methotrexate for example. Even drugs that are said to cross the BBB, such as temozolomide, the current mainstay chemotherapy drug in glioblastoma, do so only at ~20% of plasma levels. Many potentially useful drugs achieve brain tissue levels <1% of blood levels. We conclude that if careful step-wise study can establish safety, by delivering chemotherapy immediately after ECT we may open new and more effective treatment avenues for glioblastoma

Domain effects on the DNA-interactive properties of bacteriophage T4 gene 32 protein

Bacteriophage T4 gene 32 protein, a model for singlestrand specific nucleic acid-binding proteins, consists of three structurally and functionally distinct domains. We have studied the effects of the N and C domains on the protein structure and its nucleic acid-interactive properties. Although the presence of the C domain decreases the proteolytic susceptibility of the core (central) domain, quenching of the core tryptophan fluorescence by iodide is unaltered by the presence of the terminal domains. These results suggest that the overall conformation of the core domain remains largely independent of the flanking domains. Removal of the N or the C terminus does not abolish the DNA renaturation activity of the protein. However, intact protein and its three truncated forms differ in DNA helix-destabilizing activity. The C domain alone is responsible for the kinetic barrier to natural DNA helix destabilization seen with intact protein. Intact protein and core domain potentiate the DNA helix-destabilizing activity of truncated protein lacking only the C domain (*I), enhancing the observed hyperchromicity while increasing the melting temperature. Proteolysis experiments suggest that the affinity of core domain for single-stranded DNA is increased in the presence of *I. We propose that *I can “mingle” with intact protein or core domain while bound to single-stranded DNA.Journal ArticleFinal article publishe

Possible model for crotamine – double stranded DNA major groove interaction.

<p>Crotamine (1h50.pdb) and DNA (1bna.pdb) were generated using Rasmol. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048913#pone.0048913-Sayle1" target="_blank">[46]</a> Sidechains of potentially interacting arginine and tryptophan residues are indicated. The locations of the six lysine ε-NH₂ groups visible in the pictured orientation are indicated by circles.</p

Spectrophotometry of crotamine-DNA mixtures in 0.02 M Hepes, pH 7.5, 0.01 M NaCl, 0.0001 M EDTA (standard buffer with indicated [NaCl]).

<p>A. Spectra of 4.1×10⁻⁵ M ds calf thymus DNA +4.3×10⁻⁶ M crotamine. ––––––, DNA alone; · · · · ·, crotamine alone; – – – –, DNA + crotamine prior to centrifuging; – · – · – · –, DNA + crotamine supernatant after centrifuging; – · · – · · – · · –, sum of individual DNA and crotamine spectra. B. Spectra of 2.5×10⁻⁵ M(p) 25-mer (CCG)₈C and 2.5×10⁻⁶ M crotamine. ––––––, DNA alone; · · · · ·, crotamine alone; – – – –, DNA + crotamine. C. Titration of ds calf thymus DNA with crotamine; starting [DNA] = 2.22×10⁻⁵ M(p). D. Titration of d(CCG)₈C with crotamine; starting [DNA] = 1.21×10⁻⁴ M(p). E. Titration of d(CCG)₈C•G(CGG)₈ with crotamine; starting [DNA] = 6.2×10⁻⁵ M(p).</p

Binding of d(CCG)₈C to Arg-Trp-Arg-Trp-Lys-Leu-NH₂ as a function of [NaCl].

<p>A. Fluorescence titrations were performed as in Fig. 3, with the following [NaCl]: ▪, 0.01 M; ♦, 0.05 M; □, 0.075 M; ▴, 0.1 M. B. NaCl reversal: ▪, reversal of 0.01 M titration of Arg-Trp-Arg-Trp-Lys-Leu-NH₂ with d(CCG)₈C_; ◯, reversal of 0.01 titration of crotamine with d(CCG)₈C (data from Fig. 3B).</p