Unfolding and refolding of the N-terminal fragments of staphylococcal nuclease R in guanidine hydrochloride

The conformational and activity changes of a family of peptide fragments of staphylococcal nuclease R, which extend from residues -6 to 102, -6 to 110, -6 to 121, -6 to 135, and -6 to 141, during unfolding and refolding in different concentrations of guanidine hydrochloride have been studied. The studies indicate that the conformational stability in guanidine hydrochloride solution of the N-terminal fragment increases with increasing chain length, and that interaction and recognition between amino acid residues which are related to formation of the native conformation also increase with growth of the peptide chain, but such interaction becomes effective only when the polypeptide chain reaches a certain length. The changes in conformation and catalytic activity of the N-terminal fragments during unfolding and refolding demonstrate that conformational adjustments are necessary during chain elongation to generate the native conformation of a biologically active protein.     

Urea denaturation of staphylococcal nuclease monitored by Fourier transform infrared spectroscopy

Hepadnavirus invasion in woodchucks has been identified as a potent inducer of autoantibodies against asialoglycoprotein receptor (anti-ASGPR), a molecule essentially unique to hepatocytes that mediate clearance of desialylated serum proteins. We evaluated the possible pathogenetic importance of anti-ASGPR triggered by woodchuck hepatitis virus (WHV), using anti-ASGPR-reactive serum immunoglobulins (Igs) from five animals with different stages of WHV hepatitis or self-limited WHV infection and isolated woodchuck hepatocytes or HepG2 cells as targets. The results revealed that WHV-induced anti-ASGPR can specifically inhibit asialoglycoprotein recognition by both homologous and heterologous liver cells, as tested in an asialofetuin (ASFN)-binding radioassay. However, the extent of the interference significantly varied (from 85% inhibition to none) for anti-ASGPR with similar titer from different animals, indicating a high degree of heterogeneity in the ASGPR epitope specificity and in the potential biological effects of these autoantibodies. The WHV-triggered anti-ASGPR also induced complement-mediated hepatocytolysis in a microculture tetrazolium (MTT) assay, which ranged from 8.9% +/- 0.3% to 33.6% +/- 3.6% (mean +/- SD) for different animals and target cell numbers. This cytopathic effect was strictly ASGPR-specific, complement-dependent, and was not related to the anti-ASGPR ability to inhibit ligand-hepatocyte binding. Our findings indicate that among pathways by which anti-ASGPR autoimmunity could cause liver damage, hepadnavirus-induced anti-ASGPR might impair hepatocytes by both disrupting clearance of desialylated proteins and activation of the complement-mediated cytolysis. These cytopathic effects might contribute to the pathogenesis, aggravate severity, and prolong recovery from liver injury in viral hepatitis.     

Stability effects of increasing the hydrophobicity of solvent-exposed side chains in staphylococcal nuclease

A total of fifty single site surface phenylalanine substitution mutants have been made in the model protein staphylococcal nuclease. The fifty residues that were replaced with phenylalanine were chosen to give a broad sampling of solvent accessibility, secondary structure, and backbone conformations. The change in the stability of each mutant protein relative to wild type was measured by guanidine hydrochloride denaturation. These results were compared to previous results obtained when these same sites were substituted with an alanine and a glycine. By this means, changes in the stability due to the loss of interactions of the wild-type side chain can be separated from the effects of introducing the bulky, hydrophobic phenylalanine in these solvent-exposed positions. In general, our results agree with the conventional wisdom that placing a hydrophobic residue in a solvent-exposed position is destabilizing in most cases, but less destabilizing than most changes in the hydrophobic core of the protein. However, the degree to which a hydrophobic surface substitution destabilizes or stabilizes a globular protein is highly context-dependent, with some mutations being as destabilizing as those in the core. This indicates that steric and packing considerations are also important on the surface of a globular protein but generally are not as important as in the interior. No evidence for the widespread occurrence of the so-called reverse hydrophobic effect at solvent-exposed sites was found. In addition, this survey of numerous sites suggests that previous measurements of alpha-helix "propensities" often seriously underestimate the importance of the environment of the side chain.     

Interactions in nonnative and truncated forms of staphylococcal nuclease as indicated by mutational free energy changes

Several mixed disulfide variants of staphylococcal nuclease have been produced by disulfide bond formation between nuclease V23C and methane, ethane, 1-propane, 1-n-butane, and 1-n-pentane thiols. Although CD spectroscopy shows that the native state is largely unperturbed, the stability toward urea-induced unfolding is highly dependent on the nature of the group at this position, with the methyl disulfide protein being the most stable. The variant produced by modification with iodoacetic acid, however, gives a CD spectrum indicative of an unfolded polypeptide. Thiol-disulfide exchange equilibrium constants between nuclease V23C and 2-hydroxyethyl disulfide have been measured as a function of urea concentration. Because thiol-disulfide exchange and unfolding are thermodynamically linked, the effects of a mutation (disulfide exchange) can be partitioned between various conformational states. In the case of unmodified V23C and the 2-hydroxyethyl protein mixed disulfide, significant effects in the nonnative states of nuclease are observed. Truncated forms of staphylococcal nuclease are thought to be partially folded and may be good models for early folding intermediates. We have characterized a truncated form of nuclease comprised of residues 1-135 with a V23C mutation after chemical modification of the cysteine residue. High-resolution size-exclusion chromatography indicates that modification brings about significant changes in the Stokes radius of the protein, and CD spectroscopy indicates considerable differences in the amount of secondary structure present. Measurement of the disulfide exchange equilibrium constant between this truncated protein and 2-hydroxyethyl disulfide indicate significant interactions between position 23 and the rest of the protein when the urea concentration is lower than 1.5 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Compact thermally-denatured state of a staphylococcal nuclease mutant from resonance energy transfer measurements

Thermal denaturation of a staphylococcal nuclease mutant K78C, where lysine 78 is replaced by cysteine, was studied by circular dichroism (CD) and resonance energy transfer. CD spectra suggest that residual structures remain in the denatured state. Steady-state energy transfer from intrinsic tyrosines to a single and intrinsic tryptophan was measured at different temperatures. In the thermally-denatured state of K78C, there is still a substantial degree of energy transfer from tyrosine(s) to tryptophan, indicating residual structures in the denatured state. The cysteine residue in mutant K78C was labeled with a cysteine specific probe IAEDANS. Fluorescence decays of the tryptophan were measured to estimate distance distributions between Trp 140 and IAEDANS at position 78. Measurements were done as a function of temperature from 4 degrees C (native) to 65 degrees C (denatured) both with and without Ca2+ and inhibitor pdTp. Below 30 degrees C, the apparent distance distribution of both the ligand-free nuclease and the enzyme with bound pdTp can be adequately described by a Gaussian model. Above 40 degrees C, where the ligand-free nuclease but not the ternary complex begins to denature, two different populations are required to fit the data both with and without pdTp. One population has a compact structure and the other has an expanded structure. As temperature rises, the population of the expanded structure increases. At the highest temperature, the non-native compact structure is still the major form (60 to 70%). The overall thermally-denatured states of staphylococcal nuclease mutant K78C in the absence and presence of ligands are thus compact and heterogeneous.     

A monoclonal antibody selected for probing the folding of staphylococcal nuclease and its N-terminal fragments

PURPOSE: Nitric oxide (NO) is generated in mammalian tissue by the conversion of L-arginine to L-citrulline. The reaction is catalyzed by nitric oxide synthase (NOS). NO has been suggested to have a dual role in tumor biology with both antitumor and tumor promoter activity. Furthermore, it has been proposed that NO contributes to interleukin-2-induced antitumor activity. Since interleukin-2 is used in the treatment of renal cell carcinoma (RCC) it was of interest to study the NOS activity in the human kidney and in RCC and its correlation to tumor grade. Furthermore, the effect of cytokine treatment on NOS activity and the effect of NO donor application was studied in cultured cells. MATERIALS AND METHODS: The effect of cytokine treatment on NOS activity and the effect of NO donor application on cell proliferation was studied in cultured human proximal tubular cells and in RCC cell lines HN4 and HN51. NOS activity was measured by the L-arginine to L-citrulline conversion assay. RESULTS: Calcium-dependent NOS activity was found in all non-malignant kidney tissues (486+/-63 pmol. min(-1) g(-1) tissue). The activity was significantly lower in RCC (24+/-6 pmol. min(-1) g(-1) tissue) and correlated with tumor grade; thus high grade tumors showed lower activity than low grade tumors. Calcium-independent NOS activity was not detected in non-malignant kidney tissue or in RCC tissue. In cultured proximal tubular cells and RCC cell lines HN4 and HN51, cytokine treatment induced a marked increase in NOS activity and NO exerted cytostatic effects on these cell lines. Conclusions: The NOS activity was higher in non-malignant kidney tissue than in RCC tissue and was inversely correlated with tumor grade. Furthermore, cytokine treatment induced a marked increase in NOS activity and NO exerted cytostatic effects on cultured proximal tubular cells and RCC cell lines.     

Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel

In order to help determine the extent to which side chain interactions within the staphylococcal nuclease beta-barrel affect its global stability, a full set of point mutants was generated for residue 27. Intrinsic tryptophan fluorescence was monitored during solvent denaturation with guanidine hydrochloride (GuHCl) and was used to calculate DeltaGH2O unfolding and m values for each mutant. In the wild type protein, residue 27 is a tyrosine which is at the first position of a type I' beta-turn, and which participates in both hydrophobic interactions and side chain to side chain hydrogen bonding. The hydrophobicity of the mutant residue was found to be the dominant factor in determining global protein stability within this series of nuclease mutants.     

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.     

Removal of histone H1 from intact nuclei alters the digestion of nucleosome core DNA by staphylococcal nuclease

We have examined the gel profiles of staphylococcal nuclease digests of intact nuclei following different extents of removal of histone H1 by low pH. It was found that the submonomer fragment pattern (i.e. fragments less than 140 base pairs (bp) changed dramatically following removal of H1. The most striking feature of this change was a marked increase in the relative intensity of a band migrating at 102 +/- 4 bp when about 20-50% of the nuclear DNA is rendered acid soluble. All other submonomer bands decreased in relative intensity. There was no evidence for an approximately 100-bp repeat pattern accompanying the enhanced generation of the 102-bp fragment following H1 removal. This result, along with the comparisons of gel profiles for different extents of digestion, suggests that removal of histone H1 from nuclei results in an increased susceptibility of the DNA to staphylococcal nuclease at one or both ends of many of the core particles and that a strong block to further digestion occurs within these core particles resulting in the formation of a relatively stable 102-bp fragment.     

Monitoring the sizes of denatured ensembles of staphylococcal nuclease proteins: implications regarding m values, intermediates, and thermodynamics

Fluorescence and size-exclusion chromatography (SEC) are used to monitor urea denaturation of wild-type staphylococcal nuclease (SN) as well as the m+ and m- mutants A69T and V66W, respectively. It is found that the SEC partition coefficient, 1/Kd, is directly proportional to the Stokes radii of proteins. From the Stokes radii, the denatured ensembles of the three proteins are found to be highly compact in the limit of low urea concentration and expand significantly with increasing urea concentration. The m values from fluorescence-detected denaturation of the SN proteins are generally considered to reflect the relative sizes of denatured ensembles. However, the rank order of m values of the SN proteins studied do not correspond to the rank order of denatured ensemble sizes detected by 1/Kd, suggesting that m values reflect more than just surface area increases on denaturation. SEC provides two complementary ways to demonstrate the existence of intermediates in urea denaturation and illustrates that V66W undergoes a three-state transition. Fluorescence-detected urea denaturations of A69T and wt SN do not correspond with 1/Kd-detected denaturation profiles, a result that would ordinarily mean that the transitions are non-two-state. However, this interpretation fails to recognize the rapidly changing size and thermodynamic character of the denatured ensembles of these proteins both within and outside of the transition zone. The implications of the changing sizes and thermodynamic character of the denatured ensembles for SN proteins are manifold, requiring a reconsideration of the thermodynamics of proteins whose denatured ensembles behave as those of SN proteins.     

Biochemical characterization of Anabaena sp. strain PCC 7120 non-specific nuclease NucA and its inhibitor NuiA

We have established overexpression systems and purification protocols for NucA and NuiA, a sugar non-specific nuclease and its protein inhibitor from Anabaena sp. strain PCC 7120, in order to characterize these proteins in detail. CD spectroscopy revealed that NucA has a similar secondary-structure composition, 13% alpha helix and 20% beta sheet, to the related Serratia nuclease, while NuiA represents a protein with a higher alpha-helical (29%) and beta-sheet (24%) content than NucA. Denaturation experiments showed that the stabilities of NucA and NuiA are in the typical range for proteins of mesophilic organisms, NuiA with deltaG0H2O = 63.4 J x mol(-1)residue, being slightly more stable than its target NucA with delta deltaG0H2O = 46.3 J x mol(-1)residue. The nuclease requires divalent metal ions as cofactors, the optimum concentration being around 5 mM for Mn2+ or Mg2+. The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn2+ = Co2+ > Mg2+ > or = Ni2+ > or Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NucA shows a pH optimum at pH 5.5-7.5. The temperature optimum is around 35 degrees C, the activation energy was calculated to be 53 kJ mol(-1). The specific activity of the nuclease towards high molecular-mass DNA is 8.4 x 10(6) Kunitz-units x mg(-1), which means that NucA is one of the most active nucleases known. Kinetic constants for the cleavage of various DNA and RNA substrates by NucA are all in the range Km < or = 0.1 mg x ml(-1) and k(cat) approximately 1000 s(-1). As other non-specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A) x d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and inhibitor. An ultracentrifugation analysis showed that NucA and NuiA form a 1:1 complex. The interaction of NucA with NuiA was also investigated by CD spectroscopy and revealed no major conformational changes upon complex formation of the two proteins.     

On the advantage of being a dimer, a case study using the dimeric Serratia nuclease and the monomeric nuclease from Anabaena sp. strain PCC 7120

The extracellular endonucleases from Serratia marcescens and Anabaena sp. are members of a family of nonspecific endonucleases. In contrast to the monomeric Anabaena nuclease, the Serratia nuclease is a dimer of two identical subunits. To find out whether the two active sites of the Serratia nuclease function independently of each other and what the advantage of being a dimer for this enzyme might be, we produced (i) dimers in which the two subunits were cross-linked, (ii) heterodimers consisting of a wild type and an inactive mutant subunit which were also cross-linked, and (iii) monomeric variants which are unable to dimerize. The monomeric H184R variant and the cross-linked S140C variant exhibit the same activity as the wild type enzyme, while the cross-linked heterodimer with one inactive subunit shows only half of the activity of the wild type enzyme, demonstrating functional independence of the two subunits of the Serratia nuclease. On the other hand at low enzyme and substrate concentrations dimeric forms of the Serratia nuclease are relatively more active than monomeric forms or the monomeric Anabaena nuclease in cleaving polynucleotides, not, however, oligonucleotides, which is correlated with the ability of dimeric forms of the Serratia nuclease to form large enzyme-substrate networks with high molecular weight DNA and to cleave polynucleotides in a processive manner. We conclude that in the natural habitat of Serratia marcescens where the supply of nutrients may become growth limiting the dimeric nuclease can fulfil its nutritive function more efficiently than a monomeric enzyme.     

Stress and strain from family roles and work-role expectations.

Used questionnaire and interview data from 200 elementary and high school teachers (primarily 21–55 yrs old) to examine the relationship between family roles and work-role expectations and stress and strain. Results are consistent with role theory's prediction that multiple roles can lead to stressors (work overload and interrole conflict) and to symptoms of strain. Family roles were related to strain by interaction with work-role expectations so that the relation between those expectations and work overload is progressively greater for single teachers, those who were married, and those who have children. In addition, family roles were directly and negatively associated with physical strain when their relation to interrole conflict was controlled, and they were indirectly related to strain through their relation to interrole conflict. Family-role expectations seemed to reduce the amount of physical strain individuals experienced. (34 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Estimation of stabilities of staphylococcal nuclease mutants (Met32-->Ala and Met32-->Leu) using molecular dynamics/free energy perturbation

We performed molecular dynamics (MD)/free energy perturbation (FEP) calculations to reproduce the experimental free energy difference of denaturation for staphylococcal nuclease mutant Met32-->Ala (M32A) and to predict the stability of the mutant Met32-->Leu (M32L). The calculated free energy difference of denaturation for the M32A of -1.9 kcal/mol was in good agreement with the experimental value. In the M32A, a small hydrophobic core formed by three aromatic rings (Tyr-27, Phe-34, Phe-76) in a wild-type crumbled as a result of exposure to water. The van der Waals interactions in the native state of the M32A were weaker than those of the wild-type, which strongly suggests that the Met-32 is important for the stability of the enzyme. The M32L has not been available yet, but is expected to retain the small hydrophobic core. The free energy difference of denaturation for the M32L was calculated to be 1.6 kcal/mol. The MD/FEP simulation showed that the native state structure of the M32L was only slightly changed when compared with that of the wild-type. It was suggested that the M32L is more stable than the wild-type because the electrostatic interactions in the denatured state are more disadvantageous than those in the native state.     

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.     

Folding of SNase R begins early during synthesis: the conformational feature of two short N-terminal fragments of staphylococcal nuclease R

The objective was to determine why some people who are involved in minor motor vehicle accidents, without loss of consciousness, have persisting headaches and neckache, and to suggest management of these symptoms. Between 1954 and 1994, over 4400 cases were referred for medico-legal opinions. A group has been selected for discussion. During the period 1954-1966, 414 cases following closed head injuries were seen with varying periods of post traumatic amnesia (PTA) from nil to greater than 72 h. The average time between the accident and the examination was 21 months. The shortest period was 3 months and the longest 7 years. The age at the time of the accident varied from 2.5 to 72 years. The largest group fell between the ages of 20 and 40 years. The main complaints were headache, giddiness, loss of concentration and poor memory. 380 were reviewed by questionnaire after settlement of the case. 112 cases of extension/flexion injuries of the neck were seen between 1985 and 1989 and their symptoms and resolution were compared with 50 cases seen over the same period following significant head or neck injury. The results showed that the more severe the head or neck injury, the less likely were the cases to suffer symptoms of post-traumatic headaches or persisting neck symptoms. In conclusion, while 70% of minor head and neck injuries settle within a few weeks of a motor vehicle accident, about 30% continue to complain of headaches and/or neck pain. The prolonged management, extensive physiotherapy and slow court settlement lead to excessive introspection and prolongation of symptoms.     

Stress and strain localization three-dimensional modeling for particle-reinforced metal matrix composites

The ductility of particle-reinforced metal matrix composites (PR MMCs) is reduced by the localization of stress and strain, which is exacerbated by microstructural heterogeneity, especially particle clustering. Herein, the effect of particle distribution on the macroscopic and microscopic response has been studied using three distinct types of three-dimensional (3D) finite-element model: a repeating unit cell, a multiparticle model, and a clustered particle model. While the repeating unit cell model represents a cubic periodic array of particles, the multiparticle model represents a random distribution of particles contained in a cube of matrix material, and the clustered particle model represents an artificially clustered distribution of particles. These models were used to study the macroscopic tensile stress-strain response as well as the underlying stress and strain fields. The results indicate that a clustered microstructure leads to a stiffer response with more hardening than that of random and periodic microstructures. Plastic flow and hydrostatic stress localization in the matrix and maximum principal stress localization in the particles are significantly higher in the clustered microstructure. Damage is expected to initiate in the cluster regions leading to low ductility. This article is based on a presentation made in the symposium entitled “Three Dimensional Materials Science” during the 2003 MS&T ’03: Materials Science & Technology Conference 2003 in Chicago, Illinois, on November 11–12, 2003, under the auspices of the ASM/MSCTS: Materials Science Critical Technology Sector Committee and the TMS/SMD: Structural Materials Division Committee.     

Effect of staphylococcal toxin on the electrophoretic mobility and zeta-potential magnitude of BALB/c strain mouse erythrocytes

Two hours after intraperitoneal injections of the staphylococcus toxin Lh=0.18 and Lh=0.15 (4 ml/kg) to BALB/c mice there was revealed a reduction of the electrophoretic mobility and of the zeta-potential of erythrocytes. In staphylococcus intoxication the value of zeta-potential varied within greater than normal range, this pointing to the individual sensitivity of mice to the toxin. The staphylococcus toxin caused the most significant reduction of the electrophoretic mobility and of the zeta-potential of erythrocytes in male mice of the BALB/c species.