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At least 235 records · Page 13Linked to original sources
PubMed2005

Cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro.

ER-60 is a PDI family protein that has protein thiol-disulfide oxidoreductase activity. It has been shown to associate with BiP in the endoplasmic reticulum. Here, we analyzed the cooperation of ER-60 and BiP in the oxidative refolding of denatured proteins in vitro. ER-60 facilitated the refolding of 20 or 30% of denatured alpha-lactalbumin or ribonuclease B during incubation for 80 min, and these levels of nearly doubled on the addition of BiP to the reaction mixture. BiP alone could not refold denatured ribonuclease B, but could refold denatured alpha-lactalbumin a little. Enhancement of oxidative refolding of alpha-lactalbumin by ER-60 could be detected only when ER-60 was present from the start of refolding. On surface plasmon resonance analysis, ER-60 was shown to associate with BiP. The association was not influenced by ATP or ADP. Domains a and/or b' of ER-60 were implicated in the association with BiP.

Endoplasmic Reticulum Chaperone BiP↗
PubMed1992

Autoantibodies to the Ro/SSA antigen are conformation dependent. I: Anti-60 kD antibodies are mainly directed to the native protein; anti-52 kD antibodies are mainly directed to the denatured protein.

Recent studies have shown that Ro/SSA autoantigen is heterogeneous and the autoanti-Ro/SSA response is correspondingly heterogeneous. There are two isoform families; the 60 kD forms and the 52 kD forms. We studied the antigenic difference between the native and denatured Ro/SSA isoforms and found that the autoanti-Ro/SSA response to the native 60 kD antigen is quite homogeneous. All anti-Ro/SSA sera recognize the native kD antigen regardless of the reactivities to the 60 kD band on the Western blot. Surprisingly, no anti-Ro/SSA sera without anti-La/SSB reacts with the native 52 kD Ro/SSA, although sera with both precipitating anti-Ro/SSA and anti-La/SSB can immunoprecipitate the native 52 kD antigen. Anti-Ro/SSA sera exist which react exclusively with the native 60 kD Ro/SSA protein (10/43, 23%) while no anti-Ro/SSA sera have been found which react exclusively with the denatured 52 kD Ro/SSA antigen. In sera with anti-Ro/SSA precipitins alone, only antibody to the denatured 52 kD Ro/SSA molecule is found! In sera with anti-Ro/SSA and anti-U1 RNP precipitins, no antibody to either native or denatured 52 kD Ro/SSA is found, while in sera with both anti-Ro/SSA and anti-La/SSB precipitins, antibodies to both the native and denatured forms of 52 kD Ro/SSA are present. These data suggest that the anti-Ro/SSA response to the 60 kD molecule is driven by the native 60 kD Ro/SSA molecule while the molecular identification of the antigen drive in the anti-52 kD Ro/SSA response is unknown.

Antibodies, Antinuclear↗
PubMed2006

Chaperones in preventing protein denaturation in living cells and protecting against cellular stress.

A variety of cellular internal and external stress conditions can be classified as proteotoxic stresses. Proteotoxic stresses can be defined as stresses that increase the fraction of proteins that are in an unfolded state, thereby enhancing the probability of the formation of intracellular aggregates. These aggregates, if not disposed, can lead to cell death. In response to the appearance of damaged proteins, cells induce the expression of heat shock proteins. These can function as molecular chaperones to prevent protein aggregation and to keep proteins in a state competent for either refolding or degradation. Most knowledge of the function and regulation (by co-factors) of individual heat shock proteins comes from cell free studies on refolding of heat- or chemically denatured, purified proteins. Unlike the experimental situation in a test tube, cells contain multiple chaperones and co-factors often moving in and out different subcompartments that contain a variety of protein substrates at different folding states. Also, within cells folding competes with the degradative machinery. In this chapter, an overview will be provided on how the main cytosolic/nuclear chaperone Hsp70 is regulated, what is known about its interaction with other main cytosolic/nuclear chaperone families (Hsp27, Hsp90, and Hsp110), and how it may function as a molecular chaperone in living mammalian cells to protect against proteotoxic stresses.

Animals↗
PubMedDate not supplied

[Changes in heat capacity upon protein denaturation].

By the use of infrared spectroscopy it has been shown that the configurational heat capacity of hydrogen bonds represents the principal contribution into the denaturational change of heat capacity of proteins.

Hot Temperature↗
PubMed1999

Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques.

Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.

Adhesins, Bacterial↗
PubMed2001

Persistence of native-like topology in a denatured protein in 8 M urea.

Experimental methods have demonstrated that when a protein unfolds, not all of its structure is lost. Here we report measurement of residual dipolar couplings in denatured forms of the small protein staphylococcal nuclease oriented in strained polyacrylamide gels. A highly significant correlation among the dipolar couplings for individual residues suggests that a native-like spatial positioning and orientation of chain segments (topology) persists to concentrations of at least 8 molar urea. These data demonstrate that long-range ordering can occur well before a folding protein attains a compact conformation, a conclusion not anticipated by any of the standard models of protein folding.

Acrylic Resins↗
PubMedDate not supplied

[Interpretation of thermograms of protein denaturation in non-equilibrium conditions].

Data are presented concerning the effect of heating rate on the denaturation parameters of small and oligomeric globular proteins: Kunitz trypsin inhibitor from soybeans and 1,5-Ribulose Bisphosphate Carboxylase from tobacco leaves. Substantional dependence of denaturation temperature on the heating rate reflects non-equilibrium pattern of denaturation of these proteins under experimental conditions. To interpret these data a kinetic approach is proposed, which permits determination of equilibrium value of the denaturation temperature and of the constant of de- and renaturation rate. The conformation transitions in the proteins studied are shown to be relatively slow processes. Their rate is comparable to the velocity of temperature change in a calorimeter, which is the cause of non-equilibrium effects in a calorimetric experiment.

Calorimetry↗
PubMed1977

Effect of chaotropic salts and protein denaturants on the thermal stability of mouse fibroblast interferon.

Altering the aqueous environment, especially with agents that affect hydrogen bonds, markedly affects the stability of mouse L cell interferon. Low pH stabilizes interferon whereas high pH labilizes it; heavy water further enhances interferon thermostability at pH 2 but not at pH 9. Exposure to the protein denaturants, 4 M-guanidine hydrochloride and 6 M-urea, significantly decreases the activity of interferon at pH 2 and pH9; however, the residual interferon activity is relatively thermostable. Certain chaotropic salts protect interferon against thermal destruction, and in terms of effectiveness, their sequence is in the order SCN- greater than 1- larger than or equal to Cl- = CiO4- - Br- greater than NO3-. Interferon becomes more stable to heat as the NaSCN concentration is increased from 0-25 M to 2-0 M. Molecular sieve chromatography of interferon in the presence of 1-5 M-NaSCN at pH 7 shows a shift in its apparent mol. wt. from 25000 to 42000. Unlike most proteins, the unfolded conformation of interferon appears to be more stable to heat than the molecule with a smaller Stokes' radius.

Deuterium↗
PubMed1998

Excluded volume in the configurational distribution of a strongly-denatured protein.

The configurational distribution of phosphoglycerate kinase (PGK) strongly-denatured in 4 M guanidine hydrochloride solution is investigated using small-angle neutron scattering (SANS) and Monte Carlo computer simulation. It is shown that the experimental scattering profile can be represented by a random flexible chain of spheres of excess scattering density with excluded volume interactions, the best agreement being achieved when partial sphere intersection is allowed. The radius of gyration of the chain increases by a factor of 4 on denaturation, whereas the average length of segments approximately 5 residues long increases by only approximately 10%, consistent with a picture in which the large expansion on denaturation originates primarily from increased long-range flexibility of the polypeptide chain. The results provide a description of the chain statistics from which the construction of starting points for simulation studies of folding of the protein can be envisaged.

Computer Simulation↗