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PubMed1992

Conformation-dependent cleavage of staphylococcal nuclease with a disulfide-linked iron chelate.

We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.

Chromatography, High Pressure Liquid↗
PubMed1992

Truncated staphylococcal nuclease is compact but disordered.

Deletion of 13 amino acids from the carboxyl terminus of the 149-amino acid staphylococcal nuclease molecule results in a denatured, partly unfolded molecule that lacks persistent secondary structure but is compact under physiological conditions. Since the modification is a carboxyl-terminal deletion, it is argued that the state resembles a peptide emerging from the ribosome just before the complete folding pathway is initiated. In this paper, we characterize the molecule by nuclear magnetic resonance, circular dichroism, and small-angle x-ray scattering measurements. The truncated nuclease shows wild-type levels of activity in the presence of calcium and is found to fold into a native-like conformation in the presence of 3',5'-bisphospho-2'-deoxythymidine, a potent inhibitor. Thus, the truncated molecule retains the capacity to fold. Our results suggest that extensive solvent exclusion generates a compact polypeptide chain prior to the development of persistent secondary structural features as a protein folds during biosynthesis.

Circular Dichroism↗
PubMed1996

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.

Circular Dichroism↗
PubMed2001

Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis.

In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococcal nuclease cooperate unequally with Glu(75) to stabilize its structure located between the C-terminal helix and beta-barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD(222 nm). Furthermore, x-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between Glu(75) and Lys(9), Tyr(93), and His(121). In experiments using double point mutations, E73G/D77G, E73G/E75G, and E75G/D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD(222 nm) close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

Intranuclear distribution of rat liver glucocorticoid receptors by nuclease digestion in a cell-free system.

Intranuclear distribution of rat liver glucocorticoid receptors was examined. When the [3H]TA-receptor complex-bound nuclei were digested with micrococcal nuclease, the [3H]TA-receptor complex sedimented coincident with mono-, di- and trinucleosomes on a glycerol gradient. The specific activity of the [3H]TA-receptor associated with chromatin fragments in the Mg-soluble fraction was 13-fold higher than that in the Mg-insoluble fraction. In adrenalectomized rats, the nucleosome-bound [3H]TA-receptor complex was reduced, but the specific activity in the Mg-soluble fraction was almost the same as that in intact rats. Endogenous glucocorticoid receptor complex associated with mononucleosomes decreased 2 weeks after adrenalectomy. These results suggest that interaction between the glucocorticoid receptor complex and chromatin acceptor sites in intact and adrenalectomized rats occurred in the transcriptionally active chromatin regions which are sensitive to micrococcal nuclease.

Adrenalectomy↗
PubMed2002

Multiplexed screening assay for mRNA combining nuclease protection with luminescent array detection.

The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.

Anti-Inflammatory Agents↗
PubMed1985

Increased nuclease sensitivity of the human interferon-alpha 1-related genes and the interferon-beta 1 gene during induction by virus.

The chromatin structure of the human interferon (IFN) genes was evaluated during induction of human lymphoblastoid (Namalwa) cells by Sendai virus. Namalwa cells were treated with bromodeoxyuridine (BrdUrd) for 36-48 h and induced with Sendai virus for 7 h; the nuclear fraction was isolated and treated with low levels of either micrococcal nuclease or DNAse I. DNA was extracted from the nuclease-treated chromatin, restricted with Eco RI and analyzed by Southern blotting using IFN-alpha 1 and -beta 1 cDNA probes. An increase in the digestibility of the IFN-alpha 1-related genes and the IFN-beta 1 gene was observed in chromatin prepared from BrdUrd-treated, Sendai virus-induced Namalwa cells as compared with chromatin from uninduced Namalwa cells. Our results indicate that, during IFN induction in Namalwa cells by Sendai virus, the IFN genes assume a more open conformation consistent with increased transcriptional activity across these genes.

Bromodeoxyuridine↗
PubMed1983

The nuclease sensitivity of active genes.

Brief micrococcal nuclease digestion of chick embryonic red blood cells results in preferential excision and solubilization of monomer nucleosomes associated with beta-globin sequences and also 5'-sequences flanking the beta-globin gene. Both regions are DNAse-I sensitive in nuclei. Such salt-soluble nucleosomes are enriched in all four major HMG proteins but HMG1 and 2 are only weakly associated. These nucleosomes appear to have lost much of the DNAse-I sensitivity of active genes. The HMG14 and 17-containing salt-soluble nucleosomes separated by electrophoresis are not DNAse-I sensitive and contain inactive gene sequences as well as active sequences. Reconstitution of HMG proteins onto bulk nucleosomes or chromatin failed to reveal an HMG-dependent sensitivity of active genes as assayed by dot-blot hybridization and it was found that the DNAse-I sensitivity of ASV proviral sequences as assayed by dot-blot hybridization was not HMG-dependent. These results indicate that higher order chromatin structures might be responsible for nuclease sensitivity of active genes.

Animals↗
PubMed1984

The chromatin structure of the human epsilon globin gene: nuclease hypersensitive sites correlate with multiple initiation sites of transcription.

We have mapped sites in chromatin flanking the epsilon-globin gene in the K562 cell which are hypersensitive to digestion with DNAseI, micrococcal nuclease and S1 nuclease. Many of those in the 5' flanking region correspond to minor upstream transcriptional starts. However, one prominent site occurs upstream of the boundary of transcription; it maps to a region with an unusual DNA sequence. In baby hamster kidney cells stably transformed with recombinant DNA containing the human epsilon-globin gene and in Cos 7 cells transiently transfected with DNA containing the epsilon-globin gene, hypersensitive sites can be demonstrated.

Animals↗
PubMed1976

Nuclease digestion in between and within nucleosomes.

In the course of digestions of rat liver nuclei with micrococcal nuclease the size of the nucleosomal DNA is shortened by 50-60 nucleotide pairs from starting lengths of about 200, 400, 600, 800, etc. nucleotide pairs in the monomeric and oligomeric nucleosomes, respectively. Acid soluble DNA material is created relatively slowly as compared to the rate of formation of subnucleosomal material. More DNA with lengths in between the 200, 400, etc. nucleotide pairs of nucleosomal DNA is formed when digestions with micrococcal nuclease are carried out at 0 to 10 degrees C compared to 40 degrees C. With DNAase II, on the other hand, formation of a 200 nucleotide pair pattern is favoured at the low temperatures. Apparently, the accessibility of potential cleavage sites in between and within nucleosomes depends strongly on the conditions of digestion. Possible reasons for this dependence are discussed.

Animals↗
PubMed1977

Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure.

We report two new findings bearing on the "supranucleo-somal" level of the structure of the Simian Virus 40 minichromosome. I) Isolated SV40 minichromosome which contains all five histones including HI/I/ exists in solution under approximately physiological ionic conditions as a compact roughly spherical particle approximately 300 A in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiological ionic strength of solution (micron approximately 0.15). Removal of HI results in a conversion of the compact minichromosomes into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contract to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nuclease-sensitive parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter-and intranucleosomal DNA in the chronatin /2,3/ readily digests the compact form of the SV40 minichromosome. Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.

Centrifugation, Density Gradient↗
PubMed1977

Methylation of nucleosomal and nuclease sensitive DNA.

The proportion of cytosines methylated in the DNA of nucleosome oligomers and of core particles appears indistinguishable from that of total nuclear DNA from CHO cells. However the DNA in nucleoprotein which is initially released from nuclei by treatment with very low levels of micrococcal nuclease and the first 10% of material rendered acid soluble by treatment of nuclei with DNase I are enriched 2 fold in their content of 5 methylcytosine. (Cessation of hydrolysis by nuclease occurs concomitantly with precipitation of nucleosomal core particles).

Cell Line↗
PubMed1980

Analysis of DNA double- and single-strand breaks by two dimensional electrophoresis: action of micrococcal nuclease on chromatin and DNA, and degradation in vivo of lens fiber chromatin.

We describe a novel system for two dimensional electrophoresis at neutral and alkaline pH for determining the double-stranded and single-stranded lengths of DNA. With this system we analysed the mode of micrococcal nuclease digestion of DNA in cellular and SV40 viral chromatin and of supercoiled SV40 DNA. The enzyme reaction occurred in two steps : the enzyme first introduced single-strand breaks, then converted these to double-strand breaks by an adjacent cleavage on the opposite strand. Digestion of cellular chromatin DNA occurred by a similar mechanism. Chromatin fragments produced by limited micrococcal nuclease action contained many single-strand breaks, which may be important when this method is used to prepare chromatin fragments for biochemical and biophysical studies. Nucleosome monomer to tetramer produced at later stages of digestion contained few if any single-strand breaks.

Animals↗
PubMed1980

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.

Animals↗
PubMed1980

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.

Acetates↗
PubMed1996

Thermodynamics of denaturation of staphylococcal nuclease mutants: an intermediate state in protein folding.

A valuable approach to understanding the forces that maintain protein structure is to analyze the thermodynamic effects of mutations on protein folding. The folding process is most often described using an energetic model that assumes a two-state transition between the native and denatured states. However, some results obtained using this approach for mutants of the protein staphylococcal nuclease have contradicted expectations from our current understanding of protein energetics. The application of differential scanning calorimetry to a set of mutant nuclease proteins allowed us to measure directly the effects of mutations on the enthalpy and heat capacity changes of unfolding, as well as on the cooperativity. We found that most of these effects can be understood with a three-state model of folding including a distinct intermediate, but not with the two-state model. Use of a three-state instead of a two-state model leads to large differences in conclusions about the stability effects of some mutations, suggesting that reevaluation of the effects of mutations on this and other proteins may be necessary to achieve an accurate description of folding energetics. The two-state assumption commonly used in protein stability studies may be an oversimplification in many cases.

Micrococcal Nuclease↗
PubMed1995

Initial studies of the equilibrium folding pathway of staphylococcal nuclease.

Spectroscopic methods were used to examine the sequential build up of structure in the denatured state of staphylococcal nuclease. The 'free energy distance' between the native and denatured states was manipulated by altering conditions in solution (for example altering urea or glycerol concentration) and by changing the amino acid sequences. Initial studies employed a fragment of nuclease, referred to as delta 131 delta, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic resonance analysis of this fragment in solution revealed a modest quantity of dynamic structure which is native-like in character. With the addition of urea, 12 new HN peaks appeared in the 1H-15N correlation spectrum, presumably as a result of the breakdown of residual structure involving the first three beta strands. With the addition of glycerol, there was a rapid increase in the quantity of beta sheet structure detected by circular dichroism spectroscopy. At very high glycerol concentrations, an increase in helical structure became apparent. These data in addition to previously published results suggest that: (i) a beta-meander (strands beta 1-beta 2-beta 3) and the second alpha helix (alpha 2) are among the most stable local structures; (ii) the five-strand beta-barrel forms in a reaction which does not require the presence of several other native substructures; and (iii) the last step on the equilibrium folding pathway may be the formation and packing of the carboxy terminal alpha helix (alpha 3) to give the native state.

Circular Dichroism↗