muramidase and 1-anilino-8-naphthalenesulfonate

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Topics: Amyloid; Amyloid beta-Peptides; Anilino Naphthalenesulfonates; Benzothiazoles; beta 2-Microglobulin; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Hydrolysis; Muramidase; Proteolysis; Trypsin

Fluorescent probes thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) are widely used to study amyloid fibrils that accumulate in the body of patients with serious diseases, such as Alzheimer's, Parkinson's, prion diseases, etc. However, the possible effect of these probes on amyloid fibrils is not well understood. In this work, we investigated the photophysical characteristics, structure, and morphology of mature amyloid fibrils formed from two model proteins, insulin and lysozyme, in the presence of ThT and ANS. It turned out that ANS affects the secondary structure of amyloids (shown for fibrils formed from insulin and lysozyme) and their fibers clusterization (valid for lysozyme fibrils), while ThT has no such effects. These results confirm the differences in the mechanisms of these dyes interaction with amyloid fibrils. Observed effect of ANS was explained by the electrostatic interactions between the dye molecule and cationic groups of amyloid-forming proteins (unlike hydrophobic binding of ThT) that induce amyloids conformational changes. This interaction leads to weakening repulsion between positive charges of amyloid fibrils and can promote their clusterization. It was shown that when fibrillogenesis conditions and, consequently, fibrils structure is changing, as well as during defragmentation of amyloids by ultrasonication, the influence of ANS to amyloids does not change, which indicates the universality of the detected effects. Based on the obtained results, it was concluded that ANS should be used cautiously for the study of amyloid fibrils, since this fluorescence probe have a direct effect on the object of study.

Topics: Amyloid; Anilino Naphthalenesulfonates; Benzothiazoles; Binding Sites; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Insulin; Muramidase; Protein Structure, Secondary; Static Electricity

Oligomeric assemblies formed from a variety of disease-associated peptides and proteins have been strongly associated with toxicity in many neurodegenerative conditions, such as Alzheimer's disease. The precise nature of the toxic agents, however, remains still to be established. We show that prefibrillar aggregates of E22G (arctic) variant of the Abeta(1-42) peptide bind strongly to 1-anilinonaphthalene 8-sulfonate and that changes in this property correlate significantly with changes in its cytotoxicity. Moreover, we show that this phenomenon is common to other amyloid systems, such as wild-type Abeta(1-42), the I59T variant of human lysozyme and an SH3 domain. These findings are consistent with a model in which the exposure of hydrophobic surfaces as a result of the aggregation of misfolded species is a crucial and common feature of these pathogenic species.

Topics: Alzheimer Disease; Amyloid; Amyloid beta-Peptides; Anilino Naphthalenesulfonates; Cell Line; Fluorescence; Humans; Hydrophobic and Hydrophilic Interactions; Muramidase; Mutation; Peptide Fragments; Protein Folding; Protein Structure, Secondary

Modern X-ray structure analysis and advances in high-throughput robotics have allowed a significant increase in the number of conditions screened for a given sample volume. An efficient evaluation of the increased amount of crystallization trials in order to identify successful experiments is now urgently required. A novel approach is presented for the visualization of crystallization experiments using fluorescence from trace amounts of a nonspecific dye. The fluorescence images obtained strongly contrast protein crystals against other phenomena, such as precipitation and phase separation. Novel software has been developed to quantitatively evaluate the crystallization outcome based on a biophysical metric correlated with voxel protein concentration. In >1500 trials, 85.6% of the successful crystallization experiments were correctly identified, yielding a 50% reduction in the number of 'missed hits' compared with current automated approaches. The use of the method in the crystallization of three previously uncharacterized proteins from the malarial parasite Plasmodium falciparum is further demonstrated.

Topics: Anilino Naphthalenesulfonates; Animals; Crystallization; Crystallography, X-Ray; Endopeptidase K; Muramidase; Plasmodium falciparum; Software

The aggregation process of wild-type human lysozyme at pH3.0 and 60 degrees C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solvent-exposed hydrophobic patches able to bind the fluorescent probe 1-anilino-naphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the beta-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.

Topics: Anilino Naphthalenesulfonates; Biopolymers; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Humans; Microscopy, Electron, Transmission; Muramidase; Spectroscopy, Fourier Transform Infrared

A technique is described whereby the addition of low concentrations (millimolar to micromolar) of the fluorescent dye 1,8-ANS to the protein solution prior to crystallization results in crystallization experiments in which protein crystals are strongly contrasted above background artifacts when exposed to low-intensity UV radiation. As 1,8-ANS does not covalently modify the protein sample, no further handling or purification steps are necessary. The system has been tested on a wide variety of protein samples and it has been shown that the addition of 1,8-ANS has no discernible effect on the crystallization frequencies or crystallization conditions of these proteins. As 1,8-ANS interacts with a wide variety of proteins, this is proposed to be a general solution for the automated classification of protein crystallization images and the detection of protein crystals. The results also demonstrate the expected discrimination between salt and protein crystals, as well as allowing the straightforward identification of small crystals that grow in precipitate or under a protein skin.

Topics: Anilino Naphthalenesulfonates; Crystallization; Crystallography, X-Ray; Endo-1,4-beta Xylanases; Fluorescent Dyes; Insulin; Muramidase; Peptide Hydrolases; Proteins; Trypsin

The thermal unfolding of full-length human recombinant alpha-helical prion protein (alpha-PrP) in neutral pH is reversible, whereas, in the presence of the osmolyte N-trimethylamine oxide (TMAO), the protein acquires a beta-sheet structure at higher temperatures and the thermal unfolding of the protein is irreversible. Lysozyme, an amyloidogenic protein similar to prion protein, regains alpha-helical structure on cooling from its thermally unfolded form in buffer and in TMAO solutions. The thermal stability of alpha-PrP decreases, whereas that of lysozyme increases in TMAO solution. Light-scattering and turbidity values indicate that beta-sheet prion protein exists as soluble oligomers that increase thioflavin T fluorescence and bind to 1-anilino 8-naphthalene sulfonic acid (ANS). The oligomers are resistant to proteinase K digestion and during incubation for long periods they form linear amyloids>5 microm long. The comparable fluorescence polarization of the tryptophan groups and their accessibility to acrylamide in alpha-PrP and oligomers indicate that the unstructured N-terminal segments of the protein, which contain the tryptophan groups, do not associate among themselves during oligomerization. Partial unfolding of alpha-helical prion protein in TMAO solution leads to its structural conversion to misfolded beta-sheet form. The formation of the misfolded prion protein oligomers and their polymerization to amyloids in TMAO are unusual, since the osmolyte generally induces denatured protein to fold to a native-like state and protects proteins from thermal denaturation and aggregation.

Topics: Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Circular Dichroism; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Fluorescence; Hot Temperature; Humans; Hydrogen-Ion Concentration; Methylamines; Mice; Muramidase; Peptide Fragments; Prions; Protein Binding; Protein Structure, Secondary; Recombinant Proteins; Scattering, Radiation; Solubility; Thiazoles; Tryptophan

Trigger factor (TF) is an important catalyst of nascent peptide folding and possesses both peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. TF has a modular structure, containing three domains with distinct structural and functional properties. The guanidine hydrochloride (GuHCl) induced unfolding of TF was investigated by monitoring Trp fluorescence, far-UV CD, second-derivative UV absorption, enzymatic and chaperone activities, chemical crosslinking and binding of the hydrophobic dye, 1-anilinonaphthalene-8-sulfonate (ANS); and was compared to the urea induced unfolding. The native state of TF was found to bind ANS in 1:1 stoichiometry with a K(d) of 84 microM. A native-like state, N', is stable around 0.5 M GuHCl, and shows increased ANS binding, while retaining PPIase activity and most secondary and tertiary structure, but loses chaperone and dimerization activities, consistent with slight conformational rearrangement. A compact denatured state, I, is populated around 1.0 M GuHCl, is inactive and does not show significant binding to ANS. The data suggest that TF unfolds in a stepwise manner, consistent with its modular structure. The ability of TF to undergo structural rearrangement to maintain enzymatic activity while reducing chaperone and dimerization abilities may be related to the physiological function of TF.

Topics: Anilino Naphthalenesulfonates; Circular Dichroism; Cross-Linking Reagents; Dimerization; Electrophoresis, Polyacrylamide Gel; Escherichia coli Proteins; Guanidine; Molecular Chaperones; Muramidase; Peptidylprolyl Isomerase; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Spectrometry, Fluorescence; Succinimides; Thermodynamics

It is believed that denatured-reduced lysozyme rapidly forms aggregates during refolding process, which is often worked around by operating at low protein concentrations or in the presence of aggregation inhibitors. However, we found that low concentration buffer alone could efficiently suppress aggregation. Based on this finding, stable equilibrium intermediate states of denatured-reduced lysozyme containing eight free SH groups were obtained in the absence of redox reagents in buffer of low concentrations alone at neutral or mildly alkaline pH. Transition in the secondary structure of the intermediate from native-like to beta-sheet was observed by circular dichroism (CD) as conditions were varied. Dynamic light scattering and ANS-binding studies showed that the self-association accompanied the conformational change and the structure rich in beta-sheet was the intermediate state for aggregation, which could form either amyloid protofibril or amorphous aggregates under different conditions as detected by Electron Microscopy. Combining the results obtained from activity analysis, RP-HPLC and CD, we show that the activity recovery was closely related to the conformation of the refolding intermediate, and buffer of very low concentration (e.g. 10mM) alone could efficiently promote correct refolding by maintaining the native-like secondary structure of the intermediate state. This study reveals reasons for lysozyme aggregation and puts new insights into protein and inclusion body refolding.

Topics: Amyloid; Anilino Naphthalenesulfonates; Chromatography, High Pressure Liquid; Circular Dichroism; Glutathione; Hydrogen-Ion Concentration; Muramidase; Protein Binding; Protein Conformation; Protein Folding; Protein Renaturation; Sodium Chloride

Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a "phase diagram" method of fluorescence show that the conformational transition between the native state and the intermediate of MM-CK occurs in the pH range of 7.0-5.2, and the transition between the intermediate and the unfolded state of the protein occurs between pH 5.2 and 3.0. The intrinsic molar enthalpy changes for formation of the unfolded state of MM-CK induced by acid at 15.0, 25.0, 30.0, and 37.0 degrees C have been determined by ITC. A large positive molar heat capacity change of the unfolding, 8.78 kcal mol-1 K-1, at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK. Combining the results from these four methods, we conclude that the acid-induced unfolding of MM-CK follows a "three-state" model and that the intermediate state of the protein is a partially folded monomer.

Topics: Acetates; Acids; Anilino Naphthalenesulfonates; Animals; Calorimetry; Creatine Kinase; Cross-Linking Reagents; Dimerization; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glutaral; Hot Temperature; Hydrogen-Ion Concentration; Mass Spectrometry; Models, Chemical; Muramidase; Muscles; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Quaternary; Rabbits; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization; Temperature; Thermodynamics; Time Factors

The binding of 1-anilino-8-naphthalene-sulfonic acid (ANS) to various globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESI-MS). Maximal ANS binding is observed in the pH range 3-5. As many as seven species of dye-bound complexes are detected for myoglobin. Similar studies were carried out with cytochrome c, carbonic anhydrase, triosephosphate isomerase, lysozyme, alpha-lactalbumin, and bovine pancreatic trypsin inhibitor (BPTI). Strong ANS binding was observed wherever molten globule states were postulated in solution. ANS binding is not observed for lysozyme and BPTI, which have tightly folded structures in the native form. Alpha-lactalbumin, which is structurally related to lysozyme but forms a molten globule at acidic pH, exhibited ANS binding. Reduction of disulfide bonds in these proteins leads to the detection of ANS binding even at neutral pH. Binding was suppressed at very low pH (<2.5), presumably due to neutralization of the charge on the sulfonate moiety. The distribution of the relative intensities of the protein bound ANS species varies with the charge state, suggesting heterogeneity of gas phase conformations. The binding strength of these complexes was qualitatively estimated by dissociating them using enhanced nozzle skimmer potentials. The skimmer voltages also affected the lower and higher charge states of these complexes in a different manner.

Topics: Anilino Naphthalenesulfonates; Aprotinin; Carbonic Anhydrases; Cytochrome c Group; Disulfides; Lactalbumin; Muramidase; Myoglobin; Oxidation-Reduction; Protein Binding; Protein Folding; Proteins; Spectrometry, Mass, Electrospray Ionization; Triose-Phosphate Isomerase

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Animals; Apoenzymes; Calorimetry, Differential Scanning; Circular Dichroism; Crystallization; Crystallography, X-Ray; Dogs; Enzyme Stability; Milk; Molecular Sequence Data; Muramidase; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation; Protons; Thermodynamics

The molten globule state of equine lysozyme is more stable than that of alpha-lactalbumin and is stabilized by non-specific hydrophobic interactions and native-like hydrophobic interactions. We constructed a chimeric protein which is produced by replacing the flexible loop (residues 105-110) in human alpha-lactalbumin with the helix D (residues 109-114) in equine lysozyme to investigate the possible role of the helix D for the high stability and native-like packing interaction in the molten globule state of equine lysozyme. The stability of the molten globule state formed by the chimeric protein to guanidine hydrochloride-induced unfolding is the same as that of equine lysozyme and is substantially greater than that of human alpha-lactalbumin, although only six residues come from equine lysozyme. Our results also suggest that the non-native interaction in the molten globule state of alpha-lactalbumin changes to the native-like packing interaction due to helix substitution. The solvent-accessibility of the Trp residues in the molten globule state of the chimeric protein is similar to that in the molten globule state of equine lysozyme in which packing interaction around the Trp residues in the native state is partially preserved. Therefore, the helix D in equine lysozyme is one of the contributing factors to the high stability and native-like packing interaction in the molten globule state of equine lysozyme. Our results indicate that the native-like packing interaction can stabilize the rudimentary intermediate which is stabilized by the non-specific hydrophobic interactions.

Topics: Acrylamide; Amino Acid Sequence; Anilino Naphthalenesulfonates; Animals; Circular Dichroism; Enzyme Stability; Fluorescent Dyes; Guanidine; Horses; Humans; Hydrogen-Ion Concentration; Lactalbumin; Molecular Sequence Data; Muramidase; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Recombinant Fusion Proteins; Solvents; Spectrometry, Fluorescence; Thermodynamics; Tryptophan

The ANS- (1-anilino-8-naphthalene sulfonate) anion is strongly, dominantly bound to cationic groups of water-soluble proteins and polyamino acids through ion pair formation. This mode of ANS- binding, broad and pH dependent, is expressed by the quite rigorous stoichiometry of ANS- bound with respect to the available summed number of H+ titrated lysine, histidine, and arginine groups. By titration calorimetry, the integral or overall enthalpies of ANS- binding to four proteins, bovine serum albumin, lysozyme, papain, and protease omega, were arithmetic sums of individual ANS(-)-polyamino acid sidechain binding enthalpies (polyhistidine, polyarginine, polylysine), weighted by numbers of such cationic groups of each protein (additivity of binding enthalpies). ANS- binding energetics to both classes of macromolecules, cationic proteins and synthetic cationic polyamino acids, is reinforced by the organic moiety (anilinonaphthalene) of ANS-. In a much narrower range of binding, where ANS- is sometimes assumed to act as a hydrophobic probe, ANS- may become fluorescent. However, the broad overall range is sharply dependent on electrostatic, ion pair formation, where the organic sulfonate group is the major determinant of binding.

Topics: Anilino Naphthalenesulfonates; Animals; Anions; Binding Sites; Cations; Cattle; Dialysis; Fluorescent Dyes; Histidine; Hydrogen-Ion Concentration; Kinetics; Muramidase; Papain; Peptides; Polylysine; Protein Binding; Serum Albumin, Bovine

The folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single-domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two-state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N-terminal subdomain (residues 13-75), and a C-terminal subdomain (residues 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full-length variants. We propose that this acid state is comprised of an unfolded N-terminal subdomain and a loosely folded C-terminal subdomain.

Topics: Anilino Naphthalenesulfonates; Bacteriophage T4; Circular Dichroism; Cloning, Molecular; Enzyme Stability; Guanidine; Hydrogen-Ion Concentration; Models, Molecular; Muramidase; Peptide Fragments; Protein Conformation; Protein Folding; Recombinant Proteins; Spectrometry, Fluorescence; Thermodynamics

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.

Topics: Anilino Naphthalenesulfonates; Animals; Betaine; Chickens; Disulfides; Escherichia coli; Flow Injection Analysis; Fluorescent Dyes; Kinetics; Muramidase; Protein Folding; Tryptophan; Tryptophan Synthase

We have recently revealed that the intramolecular Asn-glycans promote the refolding of reductively denatured bovine pancreatic RNase B, and that extramolecular Asn-glycans of both high-mannose and complex types also markedly stimulate the oxidative refolding of RNase B and its nonglycosylated form, RNase A [Yamaguchi, H. and Uchida, M. (1996) J. Biochem. 120, 474-477; Nishimura et al. (1998) J. Biochem. 123, 516-520]. The present investigation was undertaken to see whether this function of Asn-glycans is specific to the refolding of pancreatic RNases; i.e., extramolecular Asn-glycans were examined for their effects on the oxidative refolding of hen egg white lysozyme and bovine alpha-lactalbumin by monitoring changes in activity, dynamic volume, intrinsic fluorescence, and affinity for a fluorescent probe, 1-anilino-8-naphthalenesulfonate. Asn-glycans of both high-mannose and complex types markedly stimulated the oxidative refolding of these proteins, giving similar results to those previously obtained with RNases, though differences attributable to the characteristics of individual proteins were observed in the promotive effects. Thus it seems probable that Asn-glycans generally promote the proper folding of denatured polypeptides.

Topics: Anilino Naphthalenesulfonates; Animals; Asparagine; Cattle; Chickens; Fluorescent Dyes; Kinetics; Lactalbumin; Mannose; Muramidase; Pancreas; Peptides; Polysaccharides; Protein Denaturation; Protein Folding; Ribonucleases

It was suggested long ago that the reason for the considerable increase of 8-anilino-1-naphthalene-sulfonate (8-ANS) fluorescence intensity upon the transition from aqueous to organic solvents is the dissociation of 8-ANS associates. To clarify this point the dependence of spectral properties of the dye on concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. It was shown that the increase of 8-ANS concentration leads to the changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes were observed in aqueous and organic solvents for Mg2+- and NH+4-salts of 8-anilino-1-naphthalene-sulfonateic acid and reflect the existence of self-association of the dye molecules in both media. However, the decrease in fluorescence intensity induced by the self-association of the probe molecules is too small to explain weak fluorescence of 8-ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded 8-ANS molecules upon interaction of this probe with native and molten globule proteins.

Topics: Anilino Naphthalenesulfonates; Animals; beta-Lactamases; Carbonic Anhydrases; Cattle; Fluorescent Dyes; Humans; Lactalbumin; Lactoglobulins; Magnesium; Muramidase; Protein Folding; Solvents; Spectrometry, Fluorescence; Water

Topics: Acids; Alkaloids; Allopurinol; Anilino Naphthalenesulfonates; Animals; Blood Proteins; Electrophoresis; Electrophoresis, Capillary; Glucuronates; Humans; Hydrogen-Ion Concentration; Ions; Lesch-Nyhan Syndrome; Muramidase; Nucleotides; Peptide Fragments; Phosphates; Phosphoric Diester Hydrolases; Proteins; Thioglycolates; Vinyl Chloride

The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight into the nature of intermediates detected in our recent hydrogen-exchange labeling studies [Radford, S.E., et al. (1992) Nature 358, 302-307], which were performed under the same conditions. Protection from exchange results primarily from the development of stabilizing side-chain interactions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrinsic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hydrophobic structure, and these studies have been complemented by experiments involving binding to a fluorescent hydrophobic dye 1-anilino-naphthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichorism, while development of a competent active site has been probed by binding to a competitive inhibitor bearing a fluorescent label, 4-methylumbelliferyl-N,N'-diacetyl-beta-chitobiose. The combination of these techniques has enabled us to monitor the development both of the hydrophobic core of the protein and of interactions between the two folding domains. If the behavior of the tryptophans is representative of the hydrophobic residues of the protein in general, it seems that collapse is already substantial in species formed within the first few milliseconds of refolding and is highly developed in later intermediates which nonetheless appear to lack many fixed tertiary interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Anilino Naphthalenesulfonates; Animals; Binding Sites; Binding, Competitive; Chickens; Circular Dichroism; Female; Fluorescent Dyes; Guanidine; Guanidines; Hymecromone; Iodides; Kinetics; Models, Molecular; Molecular Sequence Data; Muramidase; Oligosaccharides; Protein Folding; Protein Structure, Tertiary; Spectrometry, Fluorescence

The interaction of 1-anilino-naphthalene-8-sulphonate (ANS), a probe whose fluorescence is strongly dependent on hydrophobicity of the environment, with native lysozyme and lysozyme partially unfolded by breaking the disulphide bridges and reacting the free -SH groups with iodoacetamide, has been investigated. Monitoring the intensity of ANS fluorescence and the position of the emission maximum in the presence of native and partially unfolded lysozyme indicated that unfolding resulted in the exposure of hydrophobic sites. Hydrophobic sites could not be detected when native and partially unfolded lysozyme were denatured with urea or guanidinium chloride. Protein components of the cells export machinery like 'chaperones' associate only with partially unfolded proteins and not native, folded proteins. Hence, hydrophobic regions of proteins, exposed on partial unfolding, could be the sites of recognition by 'chaperone' proteins.

Topics: Anilino Naphthalenesulfonates; Animals; Binding Sites; Chickens; Disulfides; Fluorescent Dyes; Hydrogen-Ion Concentration; Iodoacetamide; Molecular Structure; Muramidase; Protein Denaturation; Spectrometry, Fluorescence