1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

The use of 8-anilino-1-naphthalenesulfonic acid as a reporter group molecule for circular dichroism and fluorescence measurements. The effect of stearic acid and sodium dodecylsulfate on the conformation of bovine and human serum albumin.

The fluorescence probe ANS(8-anilino-1-naphthalenesulfonic acid) was employed as a reporter group molecule for circular dichroism and fluorescence measurements in order to investigate the effects of stearic acid and sodium dodecylsulfate on the conformation of bovine and human serum albumin. Stearate as well as dodecylsulfate displaces ANS from the binding to both albumins. Besides this displacement, stearate and dodecylsulfate influence the fluorescence properties and the extrinsic Cotton effects on ANS bound to both albumins. It is suggested that the origin of these effects is a microdisorganization of the albumin structure, provoked by the binding of stearate and sodium dodecylsulfate. Each of the four extrinsic CD bands of bound ANS was influenced in a different manner by the addition of stearate and dodecylsulfate. Using the data of the fluorescence measurements and of the circular dichroism measurements it was possible to differentiate the effects of one ligand on both albumins and of both ligands on one albumin more efficiently than would have been possible using one of the two methods alone. It is suggested that the use of ANS as a reporter group molecule for fluorescence and circular dichroism measurements is a very good tool to detect small changes in the environment of ligand binding sites on protein molecules.     

8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.     

Interaction of native and modified Naja melanoleuca phospholipases A2 with the fluorescent probe 8-anilinonaphtalene-1-sulfonate

The fluorescent probe 8-anilinonaphtalene-1-sulfonate (ANS) binds at the active site of the Naja melanoleuca snake venom phospholipase A2, thus protecting the enzyme against active-site-directed chemical modification. Both hydrophobic and electrostatic interactions are involved in the binding. At pH 7.5, a binding constant of 100 microM was determined, which improved twofold upon addition of the enzymatic cofactor Ca2+. The pH dependence of the ANS binding in the absence and presence of Ca2+ ions showed a perturbation of a group with a pKa value of 5.2, which could be assigned to the carboxylate group of the Ca2+-binding ligand Asp49 at the active site of the protein. Monomeric concentrations of the substrate analog n-decylphosphocholine displace ANS from the protein, indicating again that both ligands bind at the active site. Binding studies with several modified N. melanoleuca enzymes showed that a loss of enzymatic activity on aggregated substrates was correlated with a loss of affinity for the active site bound ANS molecule. It is suggested therefore, that the fluorescent ANS probe can detect structural rearrangements at the active site, which are important for enzymatic activity.     

pH-dependent aggregation of cutinase is efficiently suppressed by 1,8-ANS

We have studied the thermal stability of the triglyceride-hydrolyzing enzyme cutinase from F. solani pisi at pH values straddling the pI (pH 8.0). At the pI, increasing the protein concentration from 5 to 80 microM decreases the apparent melting temperature by 19 degrees C. This effect vanishes at pH values more than one unit away from pI. In contrast to additives such as detergents and osmolytes, the hydrophobic fluorophore 1,8-ANS completely and saturably suppresses this effect, restoring 70% of enzymatic activity upon cooling. ANS binds strongly to native cutinase as a noncompetitive inhibitor with up to 5 ANS per cutinase molecule. Only the first ANS molecule stabilizes cutinase; however, the last 4 ANS molecules decrease Tm by up to 7 degrees C. Similar pI-dependent aggregation and suppression by ANS is observed for T. lanuginosus lipase, but not for lysozyme or porcine alpha-amylase, suggesting that this behavior is most prevalent for proteins with affinity for hydrophobic substrates and consequent exposure of hydrophobic patches. Aggregation may be promoted by a fluctuating ensemble of native-like states associating via intermolecular beta-sheet rich structures unless blocked by ANS. Our data highlight the chaperone activity of small molecules with affinity for hydrophobic surfaces and their potential application as stabilizers at appropriate stoichiometries.     

Interaction of prostaglandins and fatty acids with native and acetaldehyde-alkylated proteins

Using intrinsic and probe fluorescence, microcalorimetry and isotopic methods, the interactions of prostaglandins (PG) E2 and F2 alpha and some fatty acids with native and alkylated proteins (human serum albumin (HSA) and rat liver plasma membrane PG receptors), were studied. The fatty acid and PG interactions with human serum albumin (HSA) resulted in effective quenching of fluorescence of the probe, 1.8-anilinonaphthalene sulfonate (ANS), bound to the protein. Fatty acids competed with ANS for the binding sites; the efficiency of this process increased with an increase in the number of double bonds in the fatty acid molecule. PG induced a weaker fluorescence quenching of HSA-bound ANS and stabilized the protein molecule in a lesser degree compared to fatty acids. The sites of PG E2 and F2 alpha binding did not overlap with the sites of fatty acid binding on the HSA molecule. Nonenzymatic alkylation of HSA by acetaldehyde resulted in the abnormalities of binding sites for fatty acids and PG. Modification of the plasma membrane proteins with acetaldehyde sharply diminished the density of PG E2 binding sites without changing the association constants. Alkylation did not interfere with the parameters of PG F2 alpha binding to liver membrane proteins.     

Transition of transferrin from native to fibrillar state: An implication for amyloid-linked diseases

Human transferrin (hTF), an α/β protein, transforms from its native soluble form to proto-fibrils and amyloid fibrils at 20% TFE after prolonged incubation. This type of amyloid fibrils is observed in a number of pathological disorders. Existence of dry molten-globule state, at 5% TFE, was characterized by native-like secondary structure, Trp fluorescence and negligible ANS binding, indicating its dry interior. At 15% TFE, decrease in Trp and increase in ANS fluorescence was observed with native-like secondary structure, indicating exposure to water molecule and hence, this was referred to as Wet MG state. AFM revealed protofibrils as smaller in size howbeit amyloid fibrils were long and stiffer in morphology. Amyloid fibrils were found to possess cross-linked β-sheet, lack of tertiary contacts, as revealed by CD and ATR-FTIR, enhanced Thioflavin T fluorescence and shift in Congo red absorbance. These results showed that formation of amyloid fibrils becomes favorable when protein is destabilized in suitable conditions and non-covalent interactions, particularly intermolecular hydrogen bonding becomes prominent. Protofibrils were genotoxic in nature albeit amyloid fibrils lack this effect.     

Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1

Molecular docking and ANS-displacement experiments indicated that 8-anilinonaphthalene sulfonate (ANS) binds the hydrophobic site (H-site) in the active site of dimeric class Mu rGST M1-1. The naphthalene moiety provides most of the van der Waals contacts at the ANS-binding interface while the anilino group is able to sample different rotamers. The energetics of ANS binding were studied by isothermal titration calorimetry (ITC) over the temperature range of 5-30 degrees C. Binding is both enthalpically and entropically driven and displays a stoichiometry of one ANS molecule per subunit (or H-site). ANS binding is linked to the uptake of 0.5 protons at pH 6.5. Enthalpy of binding depends linearly upon temperature yielding a DeltaC(p) of -80+/-4 cal K(-1) mol(-1) indicating the burial of solvent-exposed nonpolar surface area upon ANS-protein complex formation. While ion-pair interactions between the sulfonate moiety of ANS and protein cationic groups may be significant for other ANS-binding proteins, the binding of ANS to rGST M1-1 is primarily hydrophobic in origin. The binding properties are compared with those of other GSTs and ANS-binding proteins.     

The major CD9 and CD81 molecular partner. Identification and characterization of the complexes

By associating with specific partner molecules and with each other, the tetraspanins are thought to assemble multimolecular complexes that may be especially relevant with respect to metastasis. We have previously identified a 135-kDa molecule (CD9P-1) as a major molecular partner of CD9 in cancer cell lines. This molecule was identified, after immunoaffinity purification and mass spectrometry analysis, as the protein encoded by the KIAA1436 gene and the human ortholog of a rat protein known as FPRP. Cross-linking experiments detected a complex of the size of CD9 plus CD9P-1, showing that these glycoproteins directly associate with each other, probably in the absence of any other molecule. The use of chimeric CD9/CD82 molecules revealed the role of the second half of CD9, comprising the large extracellular loop and the fourth transmembrane domain. CD9P-1 was also shown to form separate complexes with CD81 and with an unidentified 175-kDa molecule. It also associated with other tetraspanins under conditions maintaining tetraspanin/tetraspanin interactions. The identification of a protein strongly linked to the tetraspanin web and the production of a specific monoclonal antibody will help to further characterize the role of this "web" under physiological and pathological conditions.     

A "structural" water molecule in the family of fatty acid binding proteins

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.     

Cooperative dimerization of a stably folded protein directed by a flexible RNA in the assembly of the HIV Rev dimer–RRE stem II complex

The binding of the HIV‐1 Rev protein as an oligomer to a viral RNA element, the Rev‐response element (RRE), mediates nuclear export of genomic RNA. Assembly of the Rev–RRE ribonucleoprotein (RNP) complex is nucleated by the binding of the first Rev molecule to stem IIB of the RRE. This is followed by stepwise addition of a total of ~six Rev molecules along the RRE through a combination of RNA–protein and protein–protein interactions. RRE stem II, which forms a three‐way junction consisting of stems IIA, IIB and IIC, has been shown to bind to two Rev molecules in a cooperative manner, with the second Rev molecule binding to the junction region of stem II. The results of base substitutions at the stem II junction, and characterization of stem II junction variants selected from a randomized library showed that an “open” flexible structure is preferred for binding of the second Rev molecule, and that binding of the second Rev molecule to the junction region is not sequence‐specific. Alanine substitutions of a number of Rev amino acid residues implicated to be important for Rev folding in previous structural studies were found to result in a dramatic decrease in the binding of the second Rev molecule. These results support the model that proper folding of Rev is critical in ensuring that the flexible RRE is able to correctly position Rev molecules for specific RNP assembly, and suggests that targeting Rev folding may be effective in the inhibition of Rev function. Copyright © 2015 John Wiley & Sons, Ltd.     

Het-PDB Navi.: a database for protein-small molecule interactions

The genomes of more than 100 species have been sequenced, and the biological functions of encoded proteins are now actively being researched. Protein function is based on interactions between proteins and other molecules. One approach to assuming protein function based on genomic sequence is to predict interactions between an encoded protein and other molecules. As a data source for such predictions, knowledge regarding known protein-small molecule interactions needs to be compiled. We have, therefore, surveyed interactions between proteins and other molecules in Protein Data Bank (PDB), the protein three-dimensional (3D) structure database. Among 20,685 entries in PDB (April, 2003), 4,189 types of small molecules were found to interact with proteins. Biologically relevant small molecules most often found in PDB were metal ions, such as calcium, zinc, and magnesium. Sugars and nucleotides were the next most common. These molecules are known to act as cofactors for enzymes and/or stabilizers of proteins. In each case of interactions between a protein and small molecule, we found preferred amino acid residues at the interaction sites. These preferences can be the basis for predicting protein function from genomic sequence and protein 3D structures. The data pertaining to these small molecules were collected in a database named Het-PDB Navi., which is freely available at http://daisy.nagahama-i-bio.ac.jp/golab/hetpdbnavi.html and linked to the official PDB home page.     

ANS fluorescence. I. Effect of self-association on the spectral properties of the probe]

The dependence of spectral properties of Mg2+ and NH4+ salt of 8-anilino-1-naphthalenesulfonic acid (Mg-(ANS)2 and NH4-ANS, respectively) on the dye concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. We have shown that the increase in ANS concentrations leads to changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes, observed in aqueous and organic solvents for both salts of ANS, reflect the existence of self-association of the dye molecules. The decrease in fluorescence intensity induced by self-association of the probe molecules is too small to explain a weak fluorescence of ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded ANS molecules upon interaction of this probe with native and molten globule proteins.     

Ligand-induced self-association of human chorionic gonadotropin. Positive cooperativity in the binding of 8-anilino-1-naphthalenesulfonate.

Human chorionic gonadotropin (hCG) self-associates to form higher molecular weight species in the presence of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). Sedimentation equilibrium and fluorescence titration data have been analyzed in terms of a monomer-dimer-tetramer model in which the various oligomers have different affinities and/or capacities for the ligand. The results indicate that the ligand affinities are in the order tetramer greater than dimer greater than monomer whereas the numbers of ligand binding sites per mole of hCH are in the reverse order. Consequently, addition of ANS first shifts the equilibrium from monomer to tetramer and gives rise to positive cooperativity in the titration curves. At sufficiently high ANS concentration (approximately 0.5 mM), the equilibrium shifts back to the dimer because of its greater binding capacity. This is manifested by a second phase in the titration curve and a decrease in the polarization of ANS fluorescence. The results are discussed in terms of the general problem of ligand controlled protein association and are contrasted to results reported to the previous paper for the homolgous protein, human luteinizing hormone.     

Study of the structure of mammalian serum albumin using fluorescent probes

Some peculiarities of hydrophobic structure of serum albumine of some mammals were studied by NMR-spectroscopy, solubilization and fluorescent probes. It has been shown that the FNA probe is bound to the most hydrophobic cavities in the protein molecules, and the sizes of these regions in mammalian albumines are very close. The data obtained by ANS probe show that there exists a proportional relationship between the fluorescence intensity, the total volume of hydrophobic cavities and the quantity of "bound water". When using the ANS--Mg1/2 probe in all cases an increase of fluorescence intensity was obtained. It was concerned with the stabilizing effect of magnesium ions on the protein molecule.     

Initial binding process of the membrane insertase YidC with its substrate Pf3 coat protein is reversible

The binding of the inner membrane insertase YidC from Escherichia coli to its substrate, the Pf3 coat protein, was examined in vitro by fluorescence spectroscopy. Purified YidC protein was solubilized with the lipid-like detergent n-dodecylphosphocholine and noncovalently labeled with 1-anilino-naphthalene-8-sulfonate (ANS), whereas the Pf3 coat protein was kept in solution by the addition of 10% (v/v) isopropanol to the buffer. The binding of Pf3 coat protein was analyzed by fluorescence quenching of ANS bound to YidC. All binding curves showed a strict hyperbolic form at pH values between 9.0 and 5.0, indicating a reversible and noncooperative binding between YidC and its substrate. Analysis of the data revealed a dissociation constant K D for the binding process in the range of 1 microM. The pH profile of the K D values suggests that the binding of the Pf3 coat protein is dominated by hydrophobic interactions. The titration experiments provide strong evidence for a conformational change of the insertase upon binding a Pf3 coat protein molecule.     

Binding of the proteoglycan decorin to collagen type VI.

We have examined the interactions between the small dermatan sulfate proteoglycan decorin and collagen types I-VI using solid phase binding assays. The results of these studies showed that 125I-decorin bound most efficiently to collagen type VI in a time- and concentration-dependent manner. Furthermore, this interaction was specific and of moderately high affinity (Kd approximately 3 x 10(-7) M). Binding of decorin to collagen type VI appears to involve the decorin core protein rather than the glycosaminoglycan side chains, since the isolated core protein as well as a recombinant fusion protein containing a major segment (65%) of the human decorin core protein inhibited binding of 125I-decorin to collagen type VI. Other related proteoglycans and their respective core proteins also inhibited the binding of 125I-decorin to collagen type VI, whereas unrelated proteins and isolated glycosaminoglycan chains were without effect. In addition to decorin, collagen type II was also shown to bind to immobilized collagen type VI. Both interactions were effectively inhibited by preincubation of the immobilized collagen VI with decorin or collagen type II. These results suggested that the collagen type VI molecule has binding sites for collagen type II and decorin which are located in close proximity on the collagen type VI molecule. Possible functional roles of these interactions are discussed.     

Triple point mutation Asp10-->His, Asn101-->Asp, Arg148-->Ser in T4 phage lysozyme leads to the molten globule.

The triple amino acid replacement (Asp10-->His, Asn101-->Asp, Arg148-->Ser) in T4 phage lysozyme was carried out by site-directed mutagenesis. At acid pH (2.7) the mutant is in a conformational state with the properties of the molten globule: (i) the mutant protein molecule is essentially compact; (ii) its CD spectrum in the near UV region is drastically reduced in intensity as compared with the wild type protein spectrum; (iii) the CD spectrum in the far UV region indicates the presence of pronounced secondary structure in the mutant; (iv) unlike the wild type protein the mutant protein can bind the hydrophobic fluorescent probe, ANS.     

Molecular Dynamics Simulation of Model Lipid Membranes: Structural Effects of Impurities

Abstract

The gel to fluid phase transition or ordered to disordered phase transition observed in biological membranes are simulated by using constant energy Molecular Dynamics. The surface part of the membrane is modelled as a two-dimensional matrix formed by the head groups of the phospholipid molecules. Head molecules which are modelled as three spheres fused with three force centers, interact with each other via van der Waals and Coulomb type interactions. The -so called- impurity or foreign molecule embedded in the surface represents the protein type molecule which is present in biological membranes and control its activity. It is modelled as a pentagon having one force centers in each corner. It also interacts with the surface molecules again via van der Waals and Coulomb type interactions. The surface density is kept constant in the simulations of the systems with or without impurity. Structural and orientational changes due to impurity were observed and proved by monitoring two-dimensional order parameter. It has been shown that melting of the surface or breakage of the ordering of the surface molecules becomes easier and ordered to disordered phase transition temperature was lowered by 100 K if the impurity is present.     

ANS fluorescence: Potential to discriminate hydrophobic sites of proteins in solid states

In the current study, ANS fluorescence was established as a powerful tool to study proteins in solid-state. Silk fibroin from Bombyx mori cocoons was used as a paradigm protein. ANS incorporated into the films of silk fibroin exhibits fluorescence with two-lifetime components that can be assigned to the patches and/or cavities with distinct hydrophobicities. Decay associated spectra (DAS) of ANS fluorescence from both sites could be fit to the single log-normal component indicating their homogeneity. ANS binding sites in the protein film are specific and could be saturated by ANS titration. ANS located in the binding site that exhibits the long-lifetime fluorescence is not accessible to the water molecules and its DAS stays homogeneously broadened upon hydration of the protein film. In contrast, ANS from the sites demonstrating the short-lifetime fluorescence is accessible to water molecules. In the hydrated films, solvent-induced fluctuations produce an ensemble of binding sites with similar characters. Therefore, upon hydration, the short-lifetime DAS becomes significantly red-shifted and inhomogeneously broadened. The similar spectral features have previously been observed for ANS complexed with globular proteins in solution. The data reveal the origin of the short-lifetime fluorescence component of ANS bound to the globular proteins in aqueous solution. Findings from this study indicate that ANS is applicable to characterize dehydrated as well as hydrated protein aggregates, amyloids relevant to amyloid diseases, such as Alzheimer's, Parkinson, and prion diseases.