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At least 541 records · Page 30Linked to original sources
PubMed1997

A pentapeptide model for an early folding step in the refolding of staphylococcal nuclease: the role of its turn propensity.

Recently the folding of a staphylococcal nuclease (P117G) variant was examined with the hydrogen-deuterium (H-D) exchange technique. Many of the residues that showed significant protection are located in beta-sheet regions. About half the residues protected belong to an antiparallel beta-hairpin structure (residues 21-35) in the native structure. The beta-hairpin structure is formed by strands 2 and 3 of sheet 2 connected by the sequence 27Y KGQP31 in a type 1' reverse turn conformation with a 4-->1 hydrogen bonding between Q30 NH and Y27 C = O. We have targeted the conformational characterization of the peptide model Ac-YKGQP-NH2 with 1H two-dimensional nmr techniques in aqueous solution with a view to assessing its propensity to sample turn conformational forms and thus initiate the formation of beta-hairpin structure. Based upon the observed d alpha n (i, i + 1), d alpha n (i, i + 3), and dnn (i, i + 1) nuclear Overhauser effect connectivities, temperature coefficients for amide protons and conformational analysis with quantum mechanical perturbative configuration interaction over localized orbitals method, we conclude that the model peptide samples turn conformational forms with reduced conformational entropy. We suggest that the turn can nucleate the formation of the beta-hairpin structure in the refolding of nuclease. Observation of turn propensity for this sequence is consistent with the folding mechanism of the Greek key motif (present in Staphylococcal nuclease) proposed in the literature.

Micrococcal Nuclease↗
PubMed1982

Transmission and scanning electron microscopic studies of the human sperm chromatin decondensed by micrococcal nuclease and salt.

The human sperm chromatin was gently decondensed by treating the sperm head sequentially with micrococcal nuclease and 2 M NaCl. All histones, about 10% of DNA, and a small amount of degraded protamines were released into the soluble fraction, leaving mainly nucleoprotamines in the pellet fraction. Transmission and scanning electron microscopic studies revealed that the nucleoprotamine pellet consisted of chromatin cords of two dimensions, viz., 330- to 420-A and 650- to 1200-A thick cords laced together by very fine strands of 60- to 80-A fibers; both types of cord appeared knobby and had zig-zag patterns throughout their length. It appeared that these cords were derived from two types of sperm heads of approximately equal population; one type contained chiefly the thick cords and the other chiefly the thin cords. Further treatment of the pellet nuclease-NaCl with urea and mercaptoethanol resulted in the dissociation of the thick into the thin cords and unravelling of the thin cords into smaller sized fibers; whereas the treatment of the pellet nuclease-NaCl with DNAase I resulted in the disappearance of the 60- to 80-A fibers, and the remaining cords were chiefly of thick type together with the sperm head exoskeletons. From these results the packing order of the chromatin in human sperm was proposed.

Adult↗
PubMed1993

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.

Cold Temperature↗
PubMed1994

Thermodynamics of staphylococcal nuclease denaturation. I. The acid-denatured state.

Using high-sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower delta Cp and delta H for the denaturation reaction. The acid-denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid-denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of delta H on pH.

Calorimetry, Differential Scanning↗
PubMed1995

Proline cis-trans isomerization in staphylococcal nuclease: multi-substrate free energy perturbation calculations.

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116-Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (delta Gcis-->trans approximately 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (delta Hcis-->trans approximately delta TScis-->trans approximately 10 kcal/mol). There is evidence from X-ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112-117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substrates. By partitioning the search into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the unknown minor trans conformation.

Amino Acid Sequence↗
PubMed1995

Kinetic folding and unfolding of staphylococcal nuclease and its six mutants studied by stopped-flow circular dichroism.

Kinetics of refolding and unfolding of staphylococcal nuclease and its six mutants, each carrying single or double amino acid substitutions, are studied by stopped-flow circular dichroism measurements. A transient kinetic intermediate formed within 10 ms after refolding starts possesses a substantial part of the N-domain core beta-structure, whereas helices are formed at the later stages. The structure of the kinetic intermediate is less organized than the structure that is known to be formed by a nuclease 1-136 fragment. Only the refolding kinetics are affected by the mutations in all the mutants except two in which the mutations have changed the native structure. From this result and also from the locations of the mutation sites, the major N-terminal domain of the nuclease in the transition state of folding has a structure nearly identical to the native one. On the other hand, the minor C-terminal domain has previously been shown to be still disorganized in the transition state. The effects of the amino acid substitutions on the stability of the native and the transition states are in good agreement with the changes in the hydration free energy, expected for the corresponding amino acid replacements in the unfolded polypeptide. Since side chains of all the mutated residues are not accessible to solvent in the native structure, the result suggests that it is the unfolded state that is mainly affected by the mutations.

Circular Dichroism↗
PubMed1998

Conformational features of a truncated staphylococcal nuclease R (SNR135) and their implications for catalysis.

Conformational features of a truncated (14 amino acid residues deleted from the C-terminus) staphylococcal nuclease R (SNR135) and the ternary complex of SNR135-Ca2+-pdTp were studied using circular dichroism (CD) spectra and 1-anilinonaphthalene-8-sulfonate (ANS)-binding fluorescence spectra under different conditions. Kinetic parameters such as KDNAM, KDNAS, KCaM, KCaA, and KpdTpd of SNR135 were also determined. The results show that SNR135 contains some residual secondary structure and some tertiary structure elements as indicated by far-UV and near-UV CD spectra and that it has the ability to fold into a native-like state in the presence of pdTp and Ca2+, but there are obvious differences both in secondary structure and in tertiary structure between the SNR135-Ca2+-pdTp complex and SNase R. The unfolding curves in Gdn-HCl show that the stability of the native-like conformation of the SNR135-Ca2+-pdTp complex is much less than that of SNase R though the ligand (Ca2+, pdTp) binding increases the stability of the SNR135-Ca2+-pdTp complex to some extent. Comparison of the kinetic parameters of SNR135 with those of the full-length nuclease shows that both SNR135 and SNase R have the same value of KpdTpd and very similar values of KCaM and KCaA, but SNR135 has larger values of KDNAM and KDNAS than SNase R. Such results indicate that the C-terminal deletion for SNR135 does not greatly affect the ligand (Ca2+, pdTp) binding and decreases the binding affinity of the DNA substrate to the nuclease, implying that the amino acid residues at the ligand binding sites in SNR135 are probably arranged in a similar topology to those in SNase R and that effective binding of the DNA substrate to the enzyme needs the conformational integrity of the entire enzyme molecule. Furthermore, it is suggested that the binding sites of pdTp and DNA substrate may overlap but are not exactly the same. This paper also provides evidence obtained by monitoring ANS-binding fluorescence that the partially unfolded conformation of SNR135 is not in the molten globule state.

Calcium↗
PubMed1995

Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A.

An effective method for the determination of the activity of signal peptidase I (SPase I) of Escherichia coli is established using the hybrid protein pro-OmpA-nuclease A as substrate. Pro-OmpA-nuclease A, a hybrid secretory precursor was purified to homogeneity under denaturing conditions. When this protein was refolded, it could be quantitatively processed by purified SPase I. The Km of signal peptidase I was 0.0165 mM. The kcat was 8.73 s-1. The Km is 50 to 100 times lower than that obtained with peptide substrates indicating that SPase I has a significantly greater affinity for the protein substrate. The turnover number, kcat, is two to four orders of magnitude greater as well. Thus, the specificity constant, kcat/Km is six orders of magnitude greater with pro-OmpA-nuclease A than with peptide substrates. This is the first determination of kinetics of SPase I with a protein substrate.

Amino Acid Sequence↗
PubMed1994

Electrophoretic characterization of the denatured states of staphylococcal nuclease.

The denatured state of staphylococcal (staph) nuclease has been shown to be altered by mutations, which complicates studies of stability of the folded state and may be important for the folding process. The relative hydrodynamic volumes as a function of urea concentration of staph nuclease, and of mutant forms in which the native state was substantially destabilized, have been examined by urea gradient gel electrophoresis. The native conformation is unique in its stabilization by specific ligands, which made it possible to distinguish between intermediate electrophoretic mobilities resulting from a partly populated native conformation and from other partly folded conformations. Four distinct conformational states were observed; two native-like: (1) the native, with and without bound ligands, and (2) an altered native-ligand complex at high urea concentrations; plus two denatured states: (3) a partly folded compact conformation that unfolded co-operatively and was sensitive to mutation of residues in the beta-barrel portion of the protein, and (4) the unfolded state, which appeared from its hydrodynamic volume to be as unfolded as reduced, alkylated ribonuclease A, even at very low urea concentrations, and was not altered by single mutations. The effects of mutations on the denatured state of staph nuclease may be due to the occurrence of the partly folded conformation (3).

Calcium↗
PubMed2000

Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding.

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates. The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.

Amino Acid Substitution↗
PubMed1982

Distribution of chromatin proteins between fractions of hamster liver chromatin differing in their susceptibility to micrococcal nuclease.

Hamster liver nuclei were fractionated by digestion with micrococcal nuclease into nuclease released (SP) and nuclease resistant (PP) fractions varying in chemical composition and transcriptional activity. Electrophoretic analysis of histones from SP and PP showed no qualitative and quantitative differences. Apparently chromatin-bound protease activity can be found in both fractions. Nonhistone chromatin proteins isolated from SP and PP under mild conditions were fractionated by hydroxyapatite chromatography into NHCP1, NHCP2, NHCP3 and molecular heterogeneity and specificity were tested by SDS-polyacrylamide gel electrophoresis. The differences observed in nonhistone proteins are mainly of quantitative nature, however some specific polypeptides for SP and PP are observed.

Animals↗
PubMed2006

Recovery of nuclease produced by Lactococcus lactis using expanded bed ion exchange chromatography.

Expanded bed-ionic exchange chromatography (EB-IEC) was used for the recovery and purification of recombinant staphylococcal nuclease secreted by Lactococcus lactis. At the end of the fermentation process, the nuclease activity reached 39 U ml(-1). The EB-IEC performances were firstly evaluated with clarified culture broth. The isocratic elution with 0.5 M NaCl led to approximately 80% of nuclease recovery. Proceeding with 3-fold bed expansion resulted in a reduction of the resin capacity by a factor of 32% compared to the process in a packed bed configuration. Simplification of the early purification steps was reached by loading immediately the unclarified culture broth previously diluted to reduce conductivity. Presence of Cells did not affect the chromatography performances resulting in 55-fold purification with the same yield.

Bioreactors↗
PubMed1979

Accessibility of the ribosomal genes to micrococcal nuclease in Physarum polycephalum.

In Physarum polycephalum most genes coding for ribosomal RNA are not integrated in chromosomes, but are located in many copies in the nucleolus as plasmid-like palindromic DNA molecules. To find out whether coding sequences of rDNA are organized in a chromatin-like structure similar to that of bulk chromatin, nuclei were treated with micrococcal nuclease and DNA fragments were isolated. From bulk chromatin multimers of a basic unit of 170-180 base pairs were obtained. Nuclease fragmented DNA hybridized with labelled 19-S + 26-S rRNA was found to give the same saturation value as did unfragmented control DNA. No preferential degradation of ribosomal genes to acid soluble products was observed. A more detailed analysis of the nuclease degradation products was carried out with fragments separated by preparative gel electrophoresis. DNA eluted from the gels was hybridized in solution with labelled 19-S + 26-S rRNA. The coding sequences of rRNA were found to be degraded to approximately nucleosome size slightly more quickly than was the DNA of bulk chromatin. However, the distribution of the rDNA fragments on the gels did not coincide with the distribution of the fragments derived from bulk chromatin nucleosomes and their oligomers. The amount of rDNA in the interband regions was about intermediate between that found in the two adjacent bands. These results lead to the conclusion that the ribosomal genes, most of which are presumably active during rapid growth, are protected by proteins, probably histones. However, the ribosomal genes are present in a structure differing in some way from that of bulk chromatin.

DNA↗
PubMed1980

Replicative DNA synthesis and unscheduled DNA synthesis in permeable sarcoma cells studied by nuclease digestion.

About 20% of DNA replicated in vitro in permeable mouse ascites sarcoma cells showed higher sensitivity to staphylococcal nuclease than the sensitivity of bulk DNA, and the remaining part showed the same nuclease sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in vivo in intact cells, and close to that of DNA newly replicated in vivo in the presence of cycloheximide. Bleomycin-induced unscheduled DNA synthesis in permeable cells was highly sensitive to the nuclease. The results suggest that DNA replicated in vitro and parental nuclear protein form immature nucleosomes, probably in the same way as in vivo chromatin replication in the presence of protein synthesis inhibitors. It also appears that bleomycin-induced, unscheduled DNA synthesis occurs largely in the internucleosomal region.

Animals↗
PubMed1983

Transcription renders chromatin resistant to micrococcal nuclease digestion.

When isolated HeLa cell nuclei were preincubated under transcription conditions with excess E. coli RNA polymerase, chromatin DNA became relatively resistant to digestion by micrococcal nuclease. Quantitation of the DNA content in nuclei after enzyme digestion revealed that approximately twice as much nuclease was required to give the same levels of release of DNA fragments from transcribed as from untranscribed nuclei. Resistance increased with the amount of polymerase and with the time of preincubation. Since the resistance to nuclease was not observed in the presence of rifampicin or by preincubation without UTP, both RNA chain initiation and elongation were considered to be essential for the manifestation of resistance. However, when DNase 1 was used as a probe, such a change in chromatin DNA was not detected.

Chromatin↗
PubMed1992

Tissue-specific sensitivity of chromatin and the vitellogenin gene to micrococcal nuclease after continuous exposure of salmon (Salmo salar) to 17 beta-estradiol.

Smoltified Atlantic salmon (Salmo salar), 2 years old and weighing 217 +/- 13 g, were treated for 2 weeks with 17 beta-estradiol containing silastic capsules implanted intraperitoneally. Control fish received empty capsules. Vitellogenin, present in the blood of both groups of fish, was enhanced by estradiol treatment. Nuclei were isolated from liver, blood cells, and brain and incubated with increasing concentrations of micrococcal nuclease (EC 3.1.31.1). In liver there were more mononucleosomes as a percentage of total chromatin in hormone-treated than in control fish. Using vitellogenin cDNA as a probe the highest hybridization signals were seen when 2 to 4% of the chromatin was digested to mononucleosomes. In blood cell and brain nuclei independent of the extent of the chromatin released the hybridization signals remained low. The expression of the vitellogenin gene in immature females was potentiated by exogenous estradiol to give increased micrococcal nuclease sensitivity of the chromatin without enhancement of the hybridization level. Micrococcal nuclease digestion and hybridization of the vitellogenin gene demonstrated that the hepatic specificity of vitellogenin synthesis is manifested as structural modulations of the chromatin containing the vitellogenin gene.

Animals↗
PubMed1994

Resolution of proteins on a phenyl-Superose HR5/5 column and its application to examining the conformation homogeneity of refolded recombinant staphylococcal nuclease.

In order to examine the effect of amino acid substitutions on protein retention in hydrophobic interaction chromatography and the resolution of a phenyl-Superose HR5/5 column, two groups of staphylococcal nucleases, named Y113/W140 (wild-type), Y113W/W140 and Y113/W140F, Y113W/W140F, were produced by substituting tryptophan (W) for tyrosine (Y) at residue 113 and phenylalanine (F) for tryptophan (W) at residue 140. For each group, the proteins have the same amino acid at residue 140, but a different amino acid at residue 113. The solvent perturbation of nuclease fluorescence and 1,8-anilinoaphthalene-8-sulfonate binding studies showed that the substitutions do not change the side-chain positions of amino acids at residues 113 and 140. Chromatography of the proteins on the Phenyl-Superose HR5/5 column showed that the proteins with tryptophan at residue 113 have longer retention times than the proteins having tyrosine at residue 113; the proteins with the same amino acid at residue 113 have almost the same retention time regardless of substituting phenylalanine for tryptophan at residue 140. The studies clearly indicate that not all amino acid substitutions have an effect on protein retention; the contribution to retention of a given amino acid substitution depends on its position in a protein. Single amino acid substitutions at the exterior surface of a protein, which change the strength of hydrophobic interaction, can affect the protein retention in hydrophobic interaction chromatography. Staphylococcal nuclease and its mutants with only one amino acid difference on their surfaces can be discriminated by the phenyl-Superose column.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1992

A peptide model for proline isomerism in the unfolded state of staphylococcal nuclease.

Nuclear magnetic resonance spectroscopy has been used to investigate a synthetic peptide (YVYKPNNTHE) corresponding to residues 113 to 122 of staphylococcal nuclease. In the major folded state of the protein this region forms a type VIa beta-turn containing a cis Lys116-Pro117 peptide bond. There is, however, no evidence for any significant population of such a turn in the peptide in aqueous solution and the X-Pro bond is predominantly in the trans configuration. The peptide exhibits several well-resolved minor resonances due to the presence of a small fraction (4 +/- 2%) of the cis-proline isomer. The ratio of cis to trans isomer populations was found to be independent of temperature between 5 degrees C and 70 degrees C, indicating that delta H for the isomerism is close to zero. Using magnetization transfer techniques the rate of trans to cis interconversion was found to be 0.025(+/- 0.013) s-1 at 50 degrees C. The thermodynamics and kinetics of isomerism in the peptide are very similar to those estimated for the Lys116-Pro117 peptide bond in unfolded nuclease, suggesting that the cis-trans equilibrium in the unfolded protein is largely determined by the residues adjacent to Pro117 in the sequence. These results are consistent with previous suggestions that the cis-proline bond is stabilized late in the folding process and that the predominance of the cis form in folded nuclease is due to stabilizing interactions within the protein that give rise to a favorable enthalpy term.

Amino Acid Sequence↗