scholarly journals The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies

1976 ◽  
Vol 155 (2) ◽  
pp. 325-330 ◽  
Author(s):  
B Robson ◽  
R H. Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Tyrosine Residues ◽  
Low Concentrations ◽  
Unfolded State ◽  
Solvent Molecules ◽  
Homogeneous Preparation

1. A homogeneous preparation of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.

scholarly journals Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

1991 ◽  
Vol 277 (1) ◽  
pp. 207-211 ◽  
Author(s):  
Y Z Ma ◽  
C L Tsou
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Cross Linking ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Original Activity ◽  
Limited Region ◽  
Low Concentrations ◽  
Two Stages ◽  
Inhibited Enzyme

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

scholarly journals Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 641-646 ◽  
Author(s):  
David Snary ◽  
Adrian Allen ◽  
Roger H. Pain
Keyword(s):  
Molecular Weight ◽  
Conformational Changes ◽  
Sedimentation Coefficient ◽  
Water Structure ◽  
Water Soluble ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Gastric Mucus ◽  
Caesium Chloride ◽  
Low Concentrations

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S→33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs+and Cl−ions on water structure and the water–mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s025,w (33S→26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.

scholarly journals Subunit interactions in tyrosinase from frog epidermis in immobilized enzyme systems

1981 ◽  
Vol 197 (3) ◽  
pp. 581-589 ◽  
Author(s):  
J L Iborra ◽  
J A Ferragut ◽  
J A Lozano
Keyword(s):  
Fluorescence Spectra ◽  
Specific Activity ◽  
Guanidinium Chloride ◽  
Subunit Interactions ◽  
Low Concentrations ◽  
Tryptophan Residues ◽  
Unfolded State ◽  
The Matrix ◽  
Dopa Oxidase ◽  
Tetrameric Form

1. Frog epidermis tyrosinase was coupled to Sepharose activated with low concentrations of CNBr. The tetrameric form of the enzyme was linked to the matrix via its subunits. Dissociation of the bound active enzyme with guanidinium chloride yielded an active immobilized dimeric derivative. 2. Immobilized dimeric derivative was able to interact with soluble subunits formed transiently during renaturation. An 85% recovery of the native dopa oxidase specific activity was achieved after hybridization. 3. Fluorescence spectra of different immobilized derivatives suggested that tryptophan residues were involved in the interactions between tyrosinase subunits. 4. It is suggested that the activation of the subunits of tyrosinase involves a conformational change towards a more unfolded state, which favours a reassociation to the dimeric active state.

scholarly journals Studies of the denaturation and partial renaturation of ovalbumin

1972 ◽  
Vol 129 (3) ◽  
pp. 665-676 ◽  
Author(s):  
J. C. Holt ◽  
J. M. Creeth
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Optical Rotatory Dispersion ◽  
Rotatory Dispersion ◽  
Physical Parameters ◽  
Optical Rotatory ◽  
Guanidinium Chloride ◽  
Dispersion Parameters ◽  
Low Concentrations

1. The denaturation of ovalbumin by the reagents sodium dodecyl sulphate and guanidinium chloride was investigated, by following the changes in sedimentation velocity, optical rotatory dispersion and viscosity as a function of denaturant concentration. 2. With sodium dodecyl sulphate both the optical-rotatory-dispersion parameters a0 and b0 become more negative, the sedimentation coefficient decreases and the viscosity increases; significant differences in the denaturation profiles are observed. The change in each parameter is indicative of only limited denaturation. 3. With guanidinium chloride the transition occurs over the concentration range 1–4m: more extensive changes occur in all the physical parameters than with sodium dodecyl sulphate. The values of a0 and b0 are indicative of complete denaturation. Reduction by mercaptoethanol produces only minor further changes. 4. Renaturation was attempted from both denaturants, the removal of reagent being accomplished reversibly by controlled slow dialysis. Partial renaturation was observed, but aggregated or insoluble material was produced in both cases at relatively low concentrations of denaturant. Similar behaviour was observed with fully reduced protein in guanidinium chloride–mercaptoethanol; complete renaturation could not be brought about even at very low protein concentrations.

scholarly journals Desensitization of glutamate dehydrogenase by reaction of tyrosine residues

1969 ◽  
Vol 114 (2) ◽  
pp. 419-427 ◽  
Author(s):  
N. C. Price ◽  
G. K. Radda
Keyword(s):  
Physical Properties ◽  
Glutamate Dehydrogenase ◽  
Sedimentation Coefficient ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Tyrosine Residue ◽  
Optical Rotatory ◽  
Chemical Modifications ◽  
Tyrosine Residues ◽  
Allosteric Inhibitor

1. The reaction of glutamate dehydrogenase with N-acetylimidazole and with tetranitromethane leads to modification of tyrosine residues. 2. Modification of 1 tyrosine residue/subunit does not affect the enzymic activity but decreases the response of the enzyme to the allosteric inhibitor, GTP. 3. The physical properties of the enzyme (sedimentation coefficient and optical rotatory dispersion) remain unaltered. 4. GTP partially protects against desensitization. 5. The diminished responses of the modified enzymes to GTP are also detected by using the fluorescence of 1-anilinonaphthalene-8-sulphonate as a conformational probe. 6. Difficulties that generally arise in chemical modifications from inhomogeneous distributions of products are discussed.

scholarly journals Refolding of triose phosphate isomerase

1973 ◽  
Vol 135 (1) ◽  
pp. 165-172 ◽  
Author(s):  
Stephen G. Waley
Keyword(s):  
Half Life ◽  
Glycolytic Enzyme ◽  
Enzymic Activity ◽  
Triose Phosphate Isomerase ◽  
Guanidinium Chloride ◽  
Triose Phosphate ◽  
First Order ◽  
Rate Determining Step ◽  
Low Concentrations ◽  
High Concentrations

The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E233, took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.

scholarly journals Antibacterial Peptides Resistance in Staphylococcus aureus: Various Mechanisms and the Association with Pathogenicity

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1527
Author(s):  
Miki Kawada-Matsuo ◽  
Mi Nguyen-Tra Le ◽  
Hitoshi Komatsuzawa
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Peptides ◽  
Antibacterial Peptides ◽  
Low Concentrations ◽  
Component Systems ◽  
Two Component Systems ◽  
Resistant Strains ◽  
Resistant Mutants ◽  
High Concentrations

Staphylococcus aureus is a bacterium that mainly colonizes the nasal cavity and skin. To colonize the host, it is necessary for S. aureus to resist many antibacterial factors derived from human and commensal bacteria. Among them are the bacteria-derived antimicrobial peptides (AMPs) called bacteriocins. It was reported that some two-component systems (TCSs), which are signal transduction systems specific to bacteria, are involved in the resistance to several bacteriocins in S. aureus. However, the TCS-mediated resistance is limited to relatively low concentrations of bacteriocins, while high concentrations of bacteriocins still exhibit antibacterial activity against S. aureus. To determine whether we could obtain highly bacteriocin-resistant mutants, we tried to isolate highly nisin A-resistant mutants by exposing the cells to sub-minimum inhibitory concentrations (MICs) of nisin A. Nisin A is one of the bacteriocins produced by Lactococcus lactis and is utilized as a food preservative worldwide. Finally, we obtained highly nisin A-resistant mutants with mutations in one TCS, BraRS, and in PmtR, which is involved in the expression of pmtABCD. Notably, some highly resistant strains also showed increased pathogenicity. Based on our findings, this review provides up-to-date information on the role of TCSs in the susceptibility to antibacterial peptides. Additionally, the mechanism for high antimicrobial peptides resistance and its association with pathogenicity in S. aureus is elucidated.

scholarly journals Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

1976 ◽  
Vol 155 (3) ◽  
pp. 523-534 ◽  
Author(s):  
G Allen ◽  
J I Harris
Keyword(s):  
Conformational Changes ◽  
Bacillus Stearothermophilus ◽  
Three Dimensional ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Three Dimensional Model ◽  
Muscle Enzyme ◽  
Tyrosine Residues ◽  
Pig Muscle

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.

scholarly journals Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

1993 ◽  
Vol 291 (1) ◽  
pp. 103-107 ◽  
Author(s):  
H M Zhou ◽  
X H Zhang ◽  
Y Yin ◽  
C L Tsou
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Emission Intensity ◽  
Enzyme Inactivation ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

scholarly journals ISOLATION AND PROPERTIES OF A SURFACE ANTIGEN OF STAPHYLOCOCCUS AUREUS

1962 ◽  
Vol 115 (2) ◽  
pp. 295-311 ◽  
Author(s):  
Stephen I. Morse
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Antigen ◽  
High Titer ◽  
Amino Sugar ◽  
Intradermal Injection ◽  
Aureus Strain ◽  
Isolation And Purification ◽  
Low Concentrations ◽  
Cutaneous Hypersensitivity ◽  
High Concentrations

A technique is described for the isolation and purification of an antigen released into the culture medium by Staphylococcus aureus strain Smith. The antigen was found to be homogeneous when examined by free electrophoresis and analytic ultracentrifugation. Immunologic homogeneity was established by immunoelectrophoresis and quantitative precipitin tests using high titer antiserum prepared against the homologous organism. Chemical analysis showed that the antigen contained 70 per cent carbohydrate, of which approximately 30 to 35 per cent was believed to be glucosamine. The analytic data suggested that another amino sugar, probably carboxylated, was also present, but extreme lability of this compound to mild hydrolytic procedures has thus far precluded further identification. The remainder of the antigen was composed of alanine, glutamic acid, aspartic acid, lysine, glycine, serine, and threonine. No muramic acid was found. The chemical and physical data indicate that the antigen described herein is a previously unrecognized component of Staphylococcus aureus. The purified compound was capable of absorbing agglutinating antibody from antiserum prepared against S. aureus Smith, indicating that it was a surface component of this encapsulated staphylococcus. It is proposed that the antigen be known as the Smith surface antigen (SSA). The injection of SSA into rabbits did not produce precipitating antibodies. However, SSA did precipitate at low concentrations (0.5 µg/ml) with antiserum prepared against S. aureus Smith and one other strain of S. aureus tested. Antiserum against two other aureus strains reacted only with high concentrations of SSA. SSA did not react with S. albus antiserum or with normal sera from several animal species. Experiments are in progress to define further the distribution of SSA. Intradermal injection of small quantities of SSA into rabbits immunized with S. aureus Smith evoked a reaction of cutaneous hypersensitivity, which was maximal in 8 to 12 hours. SSA appeared to be the substance responsible for the ability of S. aureus Smith to resist engulfment by phagocytes, since absorption of Smith antiserum with SSA effectively removed opsonizing antibodies. SSA induced protection in mice against experimental staphylococcal disease. The subcutaneous injection of 0.1 µg resulted in protection against a subsequent intraperitoneal challenge with 50 to 100 LD50's of S. aureus Smith suspended in mucin. Increasing as well as decreasing the immunizing dose resulted in significantly less protection.

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