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The effect of guanidinium chloride on the behaviour of human cervical-mucus glycoproteins. Evidence for unfolding regions of ordered structure in 6M-guanidinium chloride.

ABSTRACT: The macromolecular properties of cervical-mucus glycoproteins (mucins) were studied as a function of the concentration of guanidinium chloride with conventional light-scattering, photon-correlation spectroscopy and sedimentation-velocity centrifugation. No evidence for an association of the mucins in 0.2M-NaCl as compared with 6M-guanidinium chloride was found at mucin concentrations below approx. 0.5 mg/ml. However, an increase in the frictional coefficient and in the radius of gyration occurred with increasing concentrations of guanidinium chloride, in particular between 4 M and 6 M, suggesting an expansion of the individual macromolecule. The change in the particle-scattering function is consistent with a transition from a 'stiff' random coil in 0.2 M-NaCl into a more flexible one in 6 M-guanidinium chloride. We suggest that the mucins contain regions of 'ordered' structure which can undergo a reversible 'unfolding' analogous to the behaviour of a conventional globular protein exposed to a denaturing solvent. Such regions might carry sites for specific interactions between mucins and/or be decisive for their conformation and macromolecular properties in physiological solvents.

SUBMITTER: Sheehan JK

PROVIDER: S-EPMC1144065 | biostudies-other | 1984 Jul

REPOSITORIES: biostudies-other

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Hydrodynamic properties of human cervical-mucus glycoproteins in 6M-guanidinium chloride.

Project description:Cervical mucins and fragments thereof were studied by sedimentation-velocity, rotatory viscometry and laser light-scattering performed as photon-correlation spectroscopy as well as low-angle total-intensity measurements. The Mr of the whole mucins is 10 X 10(6)-15 X 10(6), whereas fragments obtained after reduction of disulphide bonds ('subunits') have Mr 2.1 X 10(6)-2.9 X 10(6), depending on the method used. Subsequent trypsin digestion of subunits afforded glycopeptides with Mr approx. 0.4 X 10(6). The high frictional ratio for the whole mucins is interpreted as a large degree of expansion. The Stokes radius calculated from the diffusion coefficient is approx. 110nm for the whole mucins, which is in agreement with that estimated from the radius of gyration (130nm) by using the concept of the equivalent hydrodynamic sphere. The ratio of the concentration-dependence parameter for the reciprocal sedimentation coefficient (Ks) to the intrinsic viscosity ( [eta] ) for the whole mucins is 1.42, suggesting that the individual macromolecule occupies a spheroidal domain in solution. The relationship between [eta] and Mr for whole mucins, subunits and T-domains suggests that they are linear flexible macromolecules behaving as somewhat 'stiff' random coils. This conclusion is supported by the relationships between the sedimentation coefficients, the diffusion coefficients and the Mr. The hydrodynamic behaviour of the mucins is thus close to that expected for coiling macromolecules entrapping a lot of solvent, which is consistent with the postulated polymeric structure.

| S-EPMC1153186 | biostudies-other

Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride.

Project description:The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.

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Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride.

Project description:The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, ?-chymotrypsinogen A and ?-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (?G0X?D) associated with the process heat denatured (X) state ? GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ?G0N?X (native (N) state ? X state), ?G0X?D and ?G0N?D (N state ? D state), and

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Contrasting Effects of Guanidinium Chloride and Urea on the Activity and Unfolding of Lysozyme.

Project description:Cosolvents play an important role in regulating the stability and function of proteins present in the cell. We studied the role of cosolvents, urea and guanidinium chloride (GdmCl), which act as protein denaturants, in the catalytic activity and structural stability of the protein lysozyme using activity measurements, spectroscopy, and molecular dynamics simulations. We find that the activity of lysozyme increases on the addition of urea, whereas it decreases sharply on the addition of GdmCl. At low GdmCl concentrations ([GdmCl] < 4 M), the activity of lysozyme decreases, even though there is no significant perturbation in the structure of the lysozyme folded state. We find that this is due to the strong interaction of the Gdm+ ion with the residues Asp52 and Glu35, which are present in the lysozyme catalytic site. In contrast, urea interacts with Trp63 present in the loop region present near the active site of lysozyme, inducing minor conformational changes in lysozyme, which can increase the activity of lysozyme. At higher denaturant concentrations, experiments show that GdmCl completely denatures the protein, whereas the folded state is stable in the presence of urea. We further show that GdmCl denatures lysozyme with the disulfide bonds intact in the protein, whereas urea denatures the protein only when the disulfide bonds are broken using reducing agents.

| S-EPMC6644995 | biostudies-literature

Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride.

Project description:Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.

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Isolation and characterization of human cervical-mucus glycoproteins.

Project description:Mucus glycoproteins (mucins) were extracted from human cervical pregnancy mucus by 6 M-guanidinium chloride in the presence of proteinase inhibitors. Purification was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/ guanidinium chloride gradients. The purified macromolecules represented approx. 85% of the total and were devoid of nucleic acids and proteins, as judged by analytical density-gradient centrifugation, disc electrophoresis and u.v. spectroscopy. Sedimentation-velocity centrifugation revealed a single unimodal peak with S20,W 50.1S in 0.2M-NaCl and 37.0S in 6 M-guanidinium chloride. Molecular weights obtained by light-scattering were 9.7 X 10(6) and 5.9 X 10(6) in 0.2M-NaCl and 6 M-guanidinium chloride respectively. The chemical analyses were typical of those of epithelial mucins. The macromolecules contained approx. 20% (w/w) of protein, and 65% (w/w) was accounted for as carbohydrate. Serine and threonine constituted 32 mol/100 mol and proline 10 mol/100 mol of the amino acids. The major sugars found were N-acetylglucosamine (12.8%), N-acetylgalactosamine (9.7%), galactose (18.7%), sialic acid (15.0%) and fucose (7.5%).

| S-EPMC1154324 | biostudies-other

Electron microscopy of cervical, gastric and bronchial mucus glycoproteins.

Project description:Mucus glycoproteins (mucins) were obtained from human cervical and pig gastric mucus as well as from chronic-bronchitic sputum after low-shear extraction. The mucus gel was solubilized in guanidinium chloride supplemented with proteinase inhibitors, and the macromolecules were purified by using isopycnic density-gradient centrifugation. The macromolecules were spread in monolayers of benzyldimethylalkyl-ammonium chloride and studied with electron microscopy after staining with uranyl acetate and/or shadowing with platinum/carbon. The mucins appeared as flexible linear threads with lengths varying from approx. 200 nm to about 400 nm. No regularly branched or star-shaped structures were observed. The macromolecular architecture of cervical, respiratory and gastric mucins is thus similar.

| S-EPMC1147252 | biostudies-other

Structural studies on the glycoproteins from bovine cervical mucus.

Project description:The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely 'naked' peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.

| S-EPMC1185863 | biostudies-other

Unfolding and refolding of phospholipase C from Bacillus cereus in solutions of guanidinium chloride.

Project description:1. Protein-fluorescence studies indicated that phospholipase C from Bacillus cereus is denatured in solutions of guanidinium chloride. The denaturation was not thermodynamically reversible and followed biphasic kinetics. 2. Guanidinium chloride solutions released the structural Zn2+ from the enzyme and rendered all histidine residues chemically reactive. In the presence of free Zn1+ the enzyme was much more resistant to denaturation. Also, the addition for free Zn2+ to the denatured enzyme induced refolding. 3. The Zn2+-free apoenzyme was much more sensitive to guanidinium chloride than was the native enzyme and the denaturation appeared to be thermodynamically reversible. 4. Guanidinium chloride denaturation was associated with a reversible inactivation of the enzyme. Heat-inactivated, coagulated enzyme was substantially re-activated on dissolution in guanidinium chloride solutions followed by dialysis against a Zn2+-containing buffer.

| S-EPMC1186658 | biostudies-other

Electron microscopy of cervical-mucus glycoproteins and fragments therefrom. The use of colloidal gold to make visible 'naked' protein regions.

Project description:Subunits of human cervical-mucus glycoproteins obtained by reductive cleavage of whole mucins and high-Mr glycopeptides (T-domains) obtained after their trypsin digestion were studied with electron microscopy after spreading the macromolecules in a monolayer of benzyldimethylalkylammonium chloride. Subunits were observed as linear and apparently flexible particles, with number- and weight-average lengths of 390 nm and 460 nm respectively. T-domains randomly distributed on the grid have number- and weight-average lengths of 90 nm and 103 nm respectively, whereas when aligned (possibly stretched by flow) they were longer, with number-average and weight-average lengths of 150 nm and 170 nm respectively. Subunits complexed with gold appeared as segmented structures, with a distribution of inter-gold distances similar to the length distribution for the relaxed T-domains. The whole mucins had few binding sites for gold, suggesting that reduction exposes hydrophobic protein-rich regions with high affinity for gold. Most T-domains had a binding site at one end, indicating the presence of a residual protruding naked peptide region. We conclude that mucins are assembled from subunits joined end-to-end, and that each subunit consists of alternating oligosaccharide 'clusters' (approx. 100 nm) and naked peptide regions which have (after reduction) a high affinity for colloidal gold.

| S-EPMC1136627 | biostudies-other

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