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PubMed1988

Oxidatively denatured proteins are degraded by an ATP-independent proteolytic pathway in Escherichia coli.

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured proteins and protein fragments, and which may act as a "secondary antioxidant defense." We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease "La"-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which were depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic responses to oxidative stress which were indistinguishable from those observed in cells with normal ATP levels. Furthermore, the proteolytic responses to oxidative damage by menadione or H2O2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease "La," these results indicate that neither ATP nor protease "La" are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which had been oxidatively denatured (in vitro) by exposure to .OH, .OH + O2- (+O2), H2O2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45 degrees C, which inactivates protease "La," actually stimulated the degradation of oxidatively denatured proteins. Although Ca2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact cells and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8, and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.

Adenosine Triphosphate↗
PubMedDate not supplied

Is there or isn't there? The case for (and against) residual structure in chemically denatured proteins.

First raised some 60 years ago, the question of whether chemically denatured proteins are fully unfolded has, in recent years, seen significantly renewed interest. This increased attention has been spurred, in large part, by new spectroscopic and computational approaches that suggest even the most highly denatured polypeptides contain significant residual structure. In contrast, the most recent scattering results uphold the long-standing view that chemically denatured proteins adopt random coil configurations. Here we review the evidence both for and against residual structure in chemically denatured proteins, and attempt to reconcile these seemingly contradictory observations.

Protein Denaturation↗
PubMed1987

Protein damage and degradation by oxygen radicals. IV. Degradation of denatured protein.

Proteolytic degradation of oxidatively damaged [3H] bovine serum albumin [( 3H]BSA) was studied during incubation with cell-free erythrocyte extracts and a wide variety (14) of purified proteases. [3H]BSA was pretreated by exposure (60Co radiation) to the hydroxyl radical (.OH), the superoxide anion radical (O2-), or the combination of .OH + O2- + oxygen. Treated (and untreated) samples were dialyzed and then incubated with erythrocyte extract or proteases for measurements of proteolytic susceptibility (release of acid-soluble counts). Both .OH and .OH + O2- + caused severalfold increases in proteolytic susceptibility (with extract and proteases), but O2- alone had no effect. Proteolytic susceptibility reached a maximum at 15 nmol of .OH/nmol of BSA and declined thereafter. In contrast, proteolytic susceptibility was still increasing at an .OH + O2-/BSA molar ratio of 100 (50% .OH + 50% O2-). Degradation in erythrocyte extracts was conducted by a novel ATP- and Ca2+-independent pathway, with maximal activity at pH 7.8. Inhibitor profiles indicate that this pathway may involve metalloproteases and serine proteases. Comparisons of proteolytic susceptibility with multiple modifications to BSA primary, secondary, and tertiary structure revealed a high correlation (r = 0.98) with denaturation/increased hydrophobicity by low concentrations of .OH. Covalent aggregation reactions (BSA cross-linking) may explain the declining proteolytic susceptibility observed at .OH/BSA molar ratios greater than 20. Protein denaturation may also have caused the increased proteolytic susceptibility induced by .OH + O2- + O2, but no simple correlation could be obtained. Results with .OH + O2- + O2 appear to have been complicated by direct BSA fragmentation reactions involving (.OH-induced) protein radicals and oxygen. These data indicate a direct and quantitative relationship between protein damage by oxygen radicals and increased proteolytic susceptibility. Oxidative denaturation may exemplify a simple, yet effective inherent mechanism for intracellular proteolysis.

Animals↗
PubMed1991

Biophysical models of protein denaturation. I. An improvement of the model of two states.

The model of two states (native and denatured), frequently used for the description of protein denaturation, has been complemented by relations defining, on theoretical grounds, the temperature dependence of the relevant thermodynamic functions. In essence, this was achieved by assuming that the temperature dependence of Gibbs free energies of the native protein and of denaturation can be approximated, within the interval (0 degree C, 100 degrees C), by second-order partial sums of Taylor series. The improved model operates with four parameters: the temperature of denaturation, the heat capacities of the native and denatured protein at the temperature of denaturation, and the entropy of denaturation at that temperature. A theoretical treatment is included of the temperature dependence of total heat capacity, the variable recorded in the form of continuous thermograms by means of differential scanning calorimetry. Our model correctly reproduces experimental thermograms of proteins and provides for the biophysical interpretation of a number of their geometric components. Fitting procedures were complemented by a newly devised method for estimating starting values of model parameters from calorimetric data. The phenomenon of cold denaturation was also reproduced quantitatively by our model, which supplies explicit proof of the exothermal nature of this phenomenon. Finally, the relationship between temperature profiles of thermodynamic functions describing denaturation has been defined by sequences of profile magnitudes at points where the profiles intersect the temperature axis and/or cross each other. Model-derived sequences of profile magnitudes, representative of cross-point temperatures and of intervals in between, together constitute a general characteristics of denaturation, uninfluenced by differences in thermodynamic stability between protein species.

Mathematics↗
PubMed1990

Thermal analysis of bacteria by differential scanning calorimetry: relationship of protein denaturation in situ to maximum growth temperature.

Differential scanning calorimetry (DSC) was used to analyze thermal transitions in two strains of the thermophile Bacillus stearothermophilus (ATCC 12016 and WAT), the mesophile Bacillus megaterium and the psychrotroph Bacillus psychrophilus. The observed transitions, representing lipid melting and DNA and protein unfolding, are compared to the maximum growth temperature (Tmax) in each species as a means of identifying critical, thermolabile targets responsible for heat-induced inhibition of growth. A low temperature, lipid transition was detected in B. stearothermophilus and B. megaterium which varied slightly with Tmax but whose high temperature end is always 22-33 degrees C below Tmax. The transition temperature (Tm) of the main melting of DNA varies from 88 to 92 degrees C, 23-32 degrees C above Tmax. The main part of the profile representing irreversible transitions is resolvable into at least three distinct peaks and is identified primarily with protein denaturation. The onset temperature for denaturation (Tl), i.e., minimum temperature of detectable denaturation, is somewhat dependent on growth temperature (Tg). Tmax for B. stearothermophilus ATCC and WAT is 69 and 56 degrees C, respectively. For cells grown between 4 and 20 degrees C below Tmax, Tl is 2-4 degrees C lower than Tmax, demonstrating that some denaturation can be tolerated before complete inhibition of growth and suggesting that inhibition of growth is due to the denaturation of a critical protein with a Tm a few degrees above Tl or to the accumulation of denatured protein to a critical level. A similar pattern holds for B. megaterium and B. psychrophilus, except that Tmax is 48 and 32.5 degrees C (Tl = 45-46 degrees C and 30 degrees C), respectively. Thus, there is an excellent correlation between the onset of protein denaturation and maximum growth temperature for these three species of the same genus. This study also demonstrates the applicability of DSC for resolving transitions in intact cells on the basis of thermostability of cellular constituents and for obtaining an overall view of macromolecular stability.

Bacillus↗
PubMed2004

Role of nuclear protein denaturation and aggregation in thermal radiosensitization.

Hyperthermia at temperatures above 41 degrees C denatures a set of thermolabile cellular proteins located in all parts of the cell. Non-histone nuclear proteins, including those comprising the nuclear matrix, appear to be particularly thermolabile. Heating isolated nuclear matrices of Chinese hamster lung (CHL) V79 cells to 46 degrees C at 1 degree C/min results in approximately 15% denaturation. Protein unfolding during denaturation exposes buried hydrophobic residues, which increases protein-protein interactions and results in the co-aggregation of denatured thermolabile proteins and native, aggregative-sensitive nuclear proteins. This aggregated protein, the majority of which is native, is insoluble and resistant to extraction during isolation of nuclei and is responsible for the increased protein content, usually expressed as an increased protein:DNA ratio, of nuclei isolated from heated cells. A large fraction of the aggregated protein is found to be associated with the nuclear matrix, distributed throughout the fibre network and nucleolus. Three general consequences of nuclear protein denaturation and aggregation of relevance to cellular damage are: (1) protein (enzyme) inactivation, both direct inactivation of thermolabile proteins and indirect inactivation due to co-aggregation; (2) reduced accessibility and altered physical properties of DNA due to masking by aggregated protein; and (3) protein redistribution into and out of the nucleus. Functional impairment of the nucleus appears to be due to one or a combination of these basic mechanisms.

Animals↗
PubMed1990

Protein denaturation in dosage forms measured by differential scanning calorimetry.

The stability of beta-galactosidase dosage forms was studied by differential scanning calorimetry (DSC). It was found that the observed enthalpy of thermal denaturation was approximately in proportion to remaining enzyme activity, and denaturation temperature was related to protein stability. These results suggest that DSC can be used to determine native proteins in dosage forms and to clarify the factors affecting protein stability. The DSC method seems to be more convenient than conventional activity assay methods, and useful to follow protein denaturation during the manufacturing process and storage of dosage forms.

Calorimetry, Differential Scanning↗
PubMed2002

Use of dielectric properties to detect egg protein denaturation.

Changes in water and ion binding that occur during egg protein denaturation can be detected by measuring the dielectric properties. The dielectric properties of egg yolk, egg white and egg ovalbumin were tested from 25 to 105 degrees C at 11 frequencies from 300-2450 MHz. DSC was used to determine the temperature of protein denaturation. Both the dielectric constant and loss factor of egg yolk decreased due to denaturation of the protein lipovitellin. The dielectric constant increased at the initial denaturation and decreased after the complete denaturation and aggregation of egg ovalbumin in both egg white and ovalbumin.

Calorimetry, Differential Scanning↗
PubMed2001

Use of dielectric properties to detect whey protein denaturation.

Denaturation of whey proteins can be detected by the dielectric properties. The dielectric properties of 20% whey protein with 0, 5 or 15% sugar, 2% salt, at pH 4 and isolated whey proteins were measured from room temperature to 100 degrees C at 300-2450 MHz. The temperature at which a decrease in the dielectric loss factor, or increase in the dielectric constant, occurred was compared to the temperature of denaturation as determined by DSC and found to match. The change in the dielectric properties is likely caused by binding of water and/or ions that occurs during protein denaturation. For most of the samples, the dielectric loss factor showed the protein denaturation, but when ions were present due to salt or low pH, the dielectric constant showed the denaturation.

Electrochemistry↗
PubMed2006

Multiple alignment tensors from a denatured protein.

The structural content of the denatured state has yet to be fully characterized. In recent years, large residual dipolar couplings (RDCs) from denatured proteins have been observed under alignment conditions produced by bicelles and strained polyacrylamide gels. In this report, we describe efforts to extend our picture of the residual structure in denatured nuclease by measuring RDCs with multiple alignment tensors. Backbone amide 15N-1H RDCs were collected from 4 M urea for a total of eight RDC data sets. The RDCs were analyzed by singular value decomposition (SVD) to determine the number of independent alignment tensors present in the data. On the basis of the resultant singular values and propagated error estimates, it is clear that there are at least three independent alignment tensors. These three independent RDC datasets can be reconstituted as orthogonal linear combinations, (OLC)-RDC datasets, of the eight actually recorded. The first, second, and third OLC-RDC datasets are highly robust to the removal of any single experimental RDC dataset, establishing the presence of three independent alignment tensors, sampled well above the level of experimental uncertainty. The observation that the RDC data span three or more dimensions of the five-dimensional parameter space demonstrates that the ensemble average structure of denatured nuclease must be asymmetric with respect to these three orthogonal principal axes, which is not inconsistent with earlier work demonstrating that it has a nativelike topology.

Bacteriophage Pf1↗
PubMed1994

Characterization of denatured protein inducers of the heat shock (stress) response in Xenopus laevis oocytes.

In addition to thermal stress, a large variety of physical and chemical treatments are known to induce heat shock gene expression. Denatured protein, thought to result from the stress condition, has been postulated to act as the common signal. Accordingly, of three pairs of native and denatured proteins injected into Xenopus laevis oocytes, only the denatured derivatives induced expression of a reporter gene from a heat shock promoter (Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986) Science 232, 522-525). These observations are extended here. Protein denaturation per se is shown to be insufficient for heat shock induction; although reduced and carboxymethylated bovine serum albumin (rcm-BSA) and alpha-crystallin elicited a stress response, many other denatured proteins had no effect. Methylation of protein lysines, done to prevent ubiquitination, suppressed heat shock induction by rcm-BSA, but enhanced induction by alpha-crystallin. Thus, the potential for a protein to be ubiquitinated is independent of its ability to induce the stress response. Instead, aggregation distinguished the proteins that were effective stress inducers, and the formation of large aggregates correlated with the magnitude of the response. This correlation may derive in part from decreased in vivo degradation rates of the inducer proteins. An apparent requirement for stress response induction that the inducer proteins be injected directly into the oocyte nucleus may relate to this issue of in vivo stability. The dependence of the stress response on the amount of injected protein is non-linear and of a form consistent with the titration of a factor that otherwise suppresses heat shock gene expression.

Animals↗
PubMed2006

Affinity chromatography of GroEL chaperonin based on denatured proteins: role of electrostatic interactions in regulation of GroEL affinity for protein substrates.

The chaperonin GroEL of the heat shock protein family from Escherichia coli cells can bind various polypeptides lacking rigid tertiary structure and thus prevent their nonspecific association and provide for acquisition of native conformation. In the present work we studied the interaction of GroEL with six denatured proteins (alpha-lactalbumin, ribonuclease A, egg lysozyme in the presence of dithiothreitol, pepsin, beta-casein, and apocytochrome c) possessing negative or positive total charge at neutral pH values and different in hydrophobicity (affinity for a hydrophobic probe ANS). To prevent the influence of nonspecific association of non-native proteins on their interaction with GroEL and make easier the recording of the complexing, the proteins were covalently attached to BrCN-activated Sepharose. At low ionic strength (lower than 60 mM), tight binding of the negatively charged denatured proteins with GroEL (which is also negatively charged) needed relatively low concentrations (approximately 10 mM) of bivalent cations Mg2+ or Ca2+. At the high ionic strength (approximately 600 mM), a tight complex was produced also in the absence of bivalent cations. In contrast, positively charged denatured proteins tightly interacted with GroEL irrespectively of the presence of bivalent cations and ionic strength of the solution (from 20 to 600 mM). These features of GroEL interaction with positively and negatively charged denatured proteins were confirmed by polarized fluorescence (fluorescence anisotropy). The findings suggest that the affinity of GroEL for denatured proteins can be determined by the balance of hydrophobic and electrostatic interactions.

Calcium↗
PubMed1987

Studies on the physical state of water in living cells and model systems. VI. Concentration-dependent sustained volume changes of dialysis sacs containing aqueous solution of native and denatured protein, gelatin, and oxygen-containing polymers immersed in solutions of Na salt and of sugar and sugar alcohol.

In this paper we studied the volume changes of dialysis sacs containing concentrated solutions of native and denatured proteins and of oxygen-containing polymers after immersion in aqueous solution of Na-citrate, D-glucose, and sorbitol of varying concentration. The results confirm the theory of cell volume regulation: volume changes of living cells in different solutions represent a balance between the tendency of intracellular proteins -- which exist in the fully extended conformation -- to polarize, sorb, and draw into the sac or cell more water and the opposite tendency to lose water from the sac or cell created by the lower level of the solutes in the cell or sac water than in the external medium. The lower level of the solutes is the consequence of the reduced solvency of the polarized water in the sac or cell water for large and complex solutes like sugar and free amino acids. This study adds another important physico-chemical attribute of the living cell that can be duplicated by aqueous solutions of gelatin, oxygen-containing polymers like PEO and PEG as well as urea-denatured proteins but not, or only weakly so, by aqueous solutions of native proteins or SDS-denatured proteins.

Animals↗
PubMed1994

Evaluation of protein-denaturing immunoassays for avidity of immunoglobulin G to rubella virus.

Immunoassays have been recently developed that measure the avidity of IgG antibodies to complex microbial antigens and are suitable for serologic diagnosis of infectious diseases. In these avidity ELISAs, protein-denaturing agents are applied either as diluents of patient sera to prevent the immune complexing of IgG (diluting principle), or the preformed complexes are treated with the protein denaturants (eluting principle). We compared four protein denaturants previously used in such assays, in a diagnostic avidity ELISA for rubella IgG. Diethylamine, guanidine, thiocyanate, or urea were applied, by either principle at various concentrations, and thiocyanate, or urea were applied, by either principle at various concentrations, and thiocyanate at an optimum pH. Patient sera obtained recently after primary infection were distinguished from sera representing past rubella immunity by any protein denaturant tested by the eluting principle, which was superior to the diluting principle.

Antibody Affinity↗
PubMed1984

Sodium dodecyl sulfate polyacrylamide gel electrophoresis of serum after protein denaturation in the presence or absence of 2-mercaptoethanol.

Human serum proteins were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after protein denaturation in the presence or absence of 2-mercaptoethanol. Both electrophoretic techniques give characteristic and reproducible banding patterns and achieve a high degree of resolution within the limits of a one-dimensional separation. The major protein bands have been identified, and the merits of the two techniques are compared. Protein denaturation in the absence of 2-mercaptoethanol gives more discrete bands for the purpose of protein identification by maintaining the disulfide-bridge-dependent polypeptide associations characteristic of many serum proteins. However, simultaneous use of both electrophoretic techniques enhances identification by exploiting the response of an individual protein to mercaptoethanol treatment. The clinical potential of this approach for detecting protein disorders is discussed.

Animals↗