Binding of bis-ANS to Bacillus subtilis lipase: A combined computational and experimental investigation

8-Anilino-1-naphthalene sulfonate (ANS) and its covalent dimer bis-ANS are widely used for titrating hydrophobic surfaces of proteins. Interest to understand the nature of interaction of these dyes with proteins was seriously pursued. However as the techniques used in these studies varied, they often provided varied information regarding stoichiometry, binding affinity, actual binding sites etc. In the present study, we used combination of computation methods (docking and MD simulation) and experimental methods (mutations, steady-state and time-resolved fluorescence) to investigate bis-ANS interaction with Bacillus subtilis lipase. We identified seven binding sites for bis-ANS on lipase using computational docking and MD simulation and verified these data using a set of single amino acid substituted mutants. Docking and MD simulation studies indicated that the binding sites were various indentations and grooves on protein surface with hydrophobic characteristics. Both hydrophobic and ionic interactions were involved in each of these binding events. We further examine the fluorescence properties of bis-ANS bound to mutant lipases that either gained or lost a binding site. Our results indicated that neither gain nor loss of single binding site caused any change in fluorescence lifetimes (and their relative amplitudes) of mutant lipase-bound bis-ANS in comparison to that bound to wild type; hence, it suggested that nature of bis-ANS binding to each of the sites in lipase was very similar.     

Misconceptions over Förster resonance energy transfer between proteins and ANS/bis-ANS: Direct excitation dominates dye fluorescence

Our aim was to disprove the widespread misconception that Förster resonance energy transfer (FRET) is the only explanation for observing fluorescence from ANS (8-anilino-1-naphthalenesulfonic acid) and bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid, dipotassium salt) following excitation at 280 nm in the presence of protein. From ultraviolet (UV) absorption spectra and fluorescence emission spectra of bis-ANS and ANS in buffer and ethanol, direct excitation at 280 nm was found to be the dominant mechanism for the resulting dye fluorescence. Furthermore, Tyr/Trp quenching studies were performed for solutions of N-acetyl-l-tryptophanamide, heat-stressed immunoglobulin G (IgG), and bovine serum albumin (BSA) by monitoring changes in steady state fluorescence spectra and time-resolved fluorescence decays as a function of dye concentration. Stronger quenching of the intrinsic BSA and IgG fluorescence in steady state than in time-resolved fluorescence by bis-ANS and ANS pointed toward static quenching being the dominant mechanism in addition to dynamic quenching and/or FRET. In conclusion, one should consider the role of direct excitation of ANS and bis-ANS at 280 nm to ensure a proper interpretation of fluorescence signals resulting from dye-protein interactions. When ANS or bis-ANS is to be used for protein characterization, we recommend selectively exciting the dyes at the higher absorption wavelength maximum (370 or 385 nm, respectively).     

Use of a hydrophobic dye to indirectly probe the structural organization and conformational plasticity of molecules in amorphous aggregates of carbonic anhydrase

Understanding protein aggregation may hold important clues to understanding what goes wrong with protein folding in neurodegenerative disorders and in bioreactors in which proteins are overexpressed. Unfortunately, aggregates tend to be intractable to most standard methods of biochemical investigation. Thus, relatively little is even now known about the micro- and macro-structural features of aggregates. To gain insights into the thermal aggregation of a model globular protein [bovine carbonic anhydrase (BCA)], we have used spectrofluorimetry to examine the binding of a hydrophobic dye, 8-anilinonaphthalene sulfonate (ANS), to hydrophobic clusters on the protein's surface both before and after heat-induced aggregation and upon cooling. Whereas native BCA shows no surface hydrophobicity, thermally aggregated BCA displays significant hydrophobicity both in the heated state and upon cooling. The timing of the addition of ANS in the course of aggregation makes no net difference to the ANS bound; we argue that this suggests that aggregates are essentially porous. Cooling of aggregates results in a dramatic, fully reversible increase in ANS binding that cannot be explained by the temperature dependence of fluorescence quantum yield alone; we argue that the enhancement of fluorescence upon cooling indicates possible structural consolidation of unfolded regions within aggregates (akin to refolding), with the required structural reorganization being facilitated by porosity. Finally, implications of porosity in aggregates are discussed, in particular, for the possible immobilization of enzymes through fusion with aggregation-prone protein domains.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Synthesis and characterization of a peptide identified as a functional element in alphaA-crystallin

Eye lens alpha-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to alpha-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in alpha-crystallin. To investigate the mechanism of the interaction between alpha-crystallin and substrate proteins, we determined the melittin-binding sites in alpha-crystallin by cross-linking studies. Localization of melittin-binding sites in alpha-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of alphaA-crystallin and FSVNLDVK of alphaB-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPEDLTVK, having the melittin as well as bis-ANS-binding sequence of alphaA-crystallin. We show that this peptide has characteristics similar to that of alphaA-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the beta3 and beta4 region present in the alpha-crystallin domain of sHSP 16.5. We hypothesize that the alpha-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.     

Differences in the pathways of proteins unfolding induced by urea and guanidine hydrochloride: molten globule state and aggregates

It was shown that at low concentrations guanidine hydrochloride (GdnHCl) can cause aggregation of proteins in partially folded state and that fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates rather than with hydrophobic clusters on the surface of protein in molten globule state. That is why the increase in ANS fluorescence intensity is often recorded in the pathway of protein denaturation by GdnHCl, but not by urea. So what was previously believed to be the molten globule state in the pathway of protein denaturation by GdnHCl, in reality, for some proteins represents the aggregates of partially folded molecules.     

Intermediate conformation between native β-sheet and non-native α-helix is a precursor of trifluoroethanol-induced aggregation of Human Carbonic Anhydrase-II

In the present work, we examined the correlation between 2,2,2-trifluoroethanol (TFE)-induced conformational transitions of human carbonic anhydrase II (HCAII) and its aggregation propensity. Circular dichroism data indicates that protein undergoes a transition from β-sheet to α-helix on addition of TFE. The protein was found to aggregate maximally at moderate concentration of TFE at which it exists somewhere between β-sheet and α-helix, probably in extended non-native β-sheet conformation. Thioflavin-T (ThT) and Congo-Red (CR) assays along with fluorescence microscopy and transmission electron microscopy (TEM) data suggest that the protein aggregates induced by TFE possess amyloid-like features. Anilino-8-naphthalene sulfonate (ANS) binding studies reveal that the exposure of hydrophobic surface(s) was maximum in intermediate conformation. Our study suggests that the exposed hydrophobic surface and/or the disruption of the structural features protecting a β-sheet protein might be the major reason(s) for the high aggregation propensity of non-native intermediate conformation of HCAII.     

Low pH-induced conformational changes in vesicular stomatitis virus glycoprotein involve dramatic structure reorganization

Membrane fusion is the key step in the entry of enveloped animal viruses into their host cells. Fusion of vesicular stomatitis virus with membranes occurs at acidic pH and is mediated by its envelope glycoprotein, the G protein. To study the structural transitions induced by acidic pH on G protein, we have extracted the protein from purified virus by incubation with nonionic detergent. At pH 6.0, purified G protein was able to mediate fusion of either phospholipid vesicles or Vero cells in culture. Intrinsic fluorescence studies revealed that changes in the environment of Trp residues occurred as pH decreases. In the absence of lipidic membranes, acidification led to G protein aggregation, whereas protein-protein interactions were substituted by protein-lipid interactions in the presence of liposomes. 1,1'-Bis(4-aniline-5-naphthalene sulfonate) (bis-ANS) binding was utilized to probe the degree of exposure of hydrophobic regions of G protein during acidification. Bis-ANS binding was maximal at pH 6.2, suggesting that a hydrophobic segment is exposed to the medium at this pH. At pH 6.0, a dramatic decrease in bis-ANS binding was observed, probably due to loss of tridimensional structure during the conformational rearrangement. This hypothesis was confirmed by circular dichroism analysis at different pH values, which showed a great decrease in alpha-helix content at pH values close to 6.0, suggesting that a reorganization of G protein secondary structure occurs during the fusion reaction. Our results indicate that G protein undergoes dramatic structural changes at acidic pH and acquires a conformational state able to interact with the target membrane.     

Mechanism of insolubilization by a single-point mutation in alphaA-crystallin linked with hereditary human cataracts

AlphaA-crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The R49C single-point mutation in alphaA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the alphaA-R49C mutant exhibit nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90 degrees light scattering, viscosity, refractive index, and bis-ANS fluorescence of lens proteins isolated from the alphaA-R49C mouse lenses and found that the concentration of total water-soluble proteins showed a pronounced decrease in alphaA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high-molecular mass aggregated material in the void volume which still remains soluble in alphaA-R49C homozygous lens homogenates. An increased level of binding of beta- and gamma-crystallin to the alpha-crystallin fraction was observed in alphaA-R49C heterozygous and homozygous lenses but not in wild-type lenses. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to alpha-crystallin from mutant as compared with the wild-type lenses. These results suggest that the decrease in the solubility of the alphaA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of alphaA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.     

Ligand binding induces a sharp decrease in hydrophobicity of folate binding protein assessed by 1-anilinonaphthalene-8-sulphonate which suppresses self-association of the hydrophobic apo-protein

High affinity folate binding protein (FBP) regulates as a soluble protein and as a cellular receptor intracellular trafficking of folic acid, a vitamin of great importance to cell growth and division. We addressed two issues of potential importance to the biological function of FBP, a possible decrease of the surface hydrophobicity associated with the ligand-induced conformation change of FBP, and protein-inter-protein interactions involved in self-association of hydrophobic apo-FBP. The extrinsic fluorescent apolar dye 1-anilinonaphthalene-8-sulphonate (ANS) exhibited enhanced fluorescence intensity and a blueshift of emission maximum from 510-520 to 460-470nm upon addition of apo-FBP indicating binding to a strongly hydrophobic environment. Neither enhancement of fluorescence nor blueshift of ANS emission maximum occurred when folate-ligated holo-FBP replaced apo-FBP. The drastic decrease in surface hydrophobicity of holo-FBP could have bearings on the biological function of FBP since changes in surface hydrophobicity have critical effects on the biological function of receptors and transport proteins. ANS interacts with exposed hydrophobic surfaces on proteins and may thereby block and prevent aggregation of proteins (chaperone-like effect). Hence, hydrophobic interactions seemed to participate in the concentration-dependent self-association of apo-FBP which was suppressed by high ANS concentrations in light scatter measurements.     

A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.

The small heat shock proteins (sHSPs) recently have been reported to have molecular chaperone activity in vitro; however, the mechanism of this activity is poorly defined. We found that HSP18.1, a dodecameric sHSP from pea, prevented the aggregation of malate dehydrogenase (MDH) and glyceraldehyde-3-phosphate dehydrogenase heated to 45 degrees C. Under conditions in which HSP18.1 prevented aggregation of substrates, size-exclusion chromatography and electron microscopy revealed that denatured substrates coated the HSP18.1 dodecamers to form expanded complexes. SDS-PAGE of isolated complexes demonstrated that each HSP18.1 dodecamer can bind the equivalent of 12 MDH monomers, indicating that HSP18.1 has a large capacity for non-native substrates compared with other known molecular chaperones. Photoincorporation of the hydrophobic probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bis-ANS) into a conserved C-terminal region of HSP18.1 increased reversibly with increasing temperature, but was blocked by prior binding of MDH, suggesting that bis-ANS incorporates proximal to substrate binding regions and that substrate-HSP18.1 interactions are hydrophobic. We also show that heat-denatured firefly luciferase bound to HSP18.1, in contrast to heat-aggregated luciferase, can be reactivated in the presence of rabbit reticulocyte or wheat germ extracts in an ATP-dependent process. These data support a model in which sHSPs prevent protein aggregation and facilitate substrate refolding in conjunction with other molecular chaperones.     

A fluorescence study of type I and type II receptors of bone morphogenetic proteins with bis-ANS (4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid)

Crystallography studies on several members of the bone morphogenetic protein (BMP) receptors suggested that hydrophobic regions in these proteins play an important role in their structure and function. In the present study, the environment sensitive fluorescent probe 4, 4'-dianilino-1, 1'-bisnaphthyl-5, 5' disulfonic acid (bis-ANS) was used to study the hydrophobic regions of the extracellular domain of the type I and II receptors for bone morphogenetic proteins (ecBMPR-IB and ecBMPR-II). A single bis-ANS binding site per receptor molecule was found for both receptors, but the two receptors interacted with bis-ANS with distinctive characteristics. A significant shift in the emission maximum from 498 to 510 nm was detected when bis-ANS binds ecBMPR-IB, but a negligible change in the emission maximum was observed when the dye binds ecBMPR-II. Under identical reaction conditions, the maximum fluorescence intensities of the probe (I(max)) for the ecBMPR-IB and -II are 4.0 and 6.2 x 10(4) arbitrary units, respectively. The probe binds to ecBMPR-IB and -II with K(d)=11.0 and 17.5 microM, respectively. The bis-ANS modified site on both receptor types was not readily accessible to acrylamide quenching. Fluorescence energy transfer experiments further revealed close proximity between the tyrosine (in ecBMPR-IB) and the tryptophan residue (in ecBMPR-II) and the respective bis-ANS binding site in these receptors. The binding of bis-ANS did not alter the ligand binding activity of ecBMPR-IB, but enhanced that of ecBMPR-II. These results show that the bis-ANS-modified hydrophobic site on the ecBMPR-IB and -II molecules plays a different functional role.     

Functional mode of NtHSP17.6, a cytosolic small heat-shock protein fromNicotiana tabacum

Small heat-shock proteins (sHsps) are ubiquitous stress proteins with molecular chaperone activity. They share characteristic homology with the α-crystallin protein of the mammalian eye lens as well as being ATP-independent in their chaperone activity. We isolated a clone for a cytosolic class I sHsp,NtHSP17.6, fromNicotiana tabacum, and analyzed its functional mode for such activity. Following its transformation intoEscherichia coli and its over-expression, NtHSPI 7.6 was purified and examinedin vitro. This purified NtHSPI 7.6 exhibited typical chaperone activity in a light-scattering test. It was enable to protect a model substrate, firefly luciferase, from heat-induced aggregation. Non-denaturing PAGE showed that NtHSP17.6 formed a dodecamer in its native conformation, and was bound to its substrate under heat stress. A labeling test with bis-ANS indicated that this binding might be linked to newly exposed hydrophobic sites of the NtHSPI 7.6 complexes during heat shock. Based on these data, we suggest that NtHSP17.6 is a molecular chaperone that functions as a dodecamer in a heat-induced manner.     

The initiation codon AUG binds at a hydrophobic site on yeast 40S ribosomal subunits as revealed by fluorescence studies with bis (1,8-anilinonaphthalenesulfonate).

Binding studies of yeast 40S ribosome with bis (1,8-anilinonaphthalenesulfonate) (bis-ANS) revealed the binding of 3-4 molecules of bis-ANS per ribosome with a dissociation constant (Kd) of 1.45 microM. Binding of AUG to the 40S subunits resulted in a concentration-dependent decrease in the bis-ANS fluorescence without displacing all of the bound bis-ANS from the ribosomes. The residual bis-ANS fluorescence at saturation with AUG corresponds to about 3 molecules of bis-ANS per ribosome. Thus AUG displaces one of the bound bis-ANS molecules. The data suggest that AUG binds at a hydrophobic site on the yeast 40S subunit.     

A thermodynamic characterization of the interaction of 8‐anilino‐1‐naphthalenesulfonic acid with native globular proteins: the effect of the ligand dimerization in the analysis of the binding isotherms

8‐Anilino‐1‐naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl‐SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl‐SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS. Copyright © 2010 John Wiley & Sons, Ltd.     

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.     

Equilibrium unfolding and conformational plasticity of troponin I and T.

The structures and stabilities of recombinant chicken muscle troponin I (TnI) and T (TnT) were investigated by a combination of bis-ANS binding and equilibrium unfolding studies. Unlike most folded proteins, isolated TnI and TnT bind the hydrophobic fluorescent probe bis-ANS, indicating the existence of solvent-exposed hydrophobic domains in their structures. Bis-ANS binding to binary or ternary mixtures of TnI, TnT and troponin C (TnC) in solution is significantly lower than binding to the isolated subunits, which can be explained by burial of previously exposed hydrophobic domains upon association of the subunits to form the native troponin complex. Equilibrium unfolding studies of TnT and TnI by guanidine hydrochloride and urea monitored by changes in far-UV CD and bis-ANS fluorescence revealed noncooperative folding transitions for both proteins and the existence of partially folded intermediate states. Taken together, these results indicate that isolated TnI and TnT are partially unstructured proteins, and suggest that conformational plasticity of the isolated subunits may play an important role in macromolecular recognition for the assembly of the troponin complex.     

Interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and human matrix metalloproteinase 7 (MMP-7) as examined by MMP-7 activity and ANS fluorescence

Human matrix metalloproteinase 7 (MMP-7) is the smallest matrix metalloproteinase. It plays important roles in tumour invasion and metastasis. 8-Anilinonaphthalene 1-sulphonate (ANS) is a fluorescent probe widely used for the analysis of proteins. It emits large fluorescence energy when its anilinonaphthalene group binds with hydrophobic regions of protein. In this study, we analysed the interaction of ANS and MMP-7. At pH 4.5-9.5, ANS inhibited MMP-7 activity in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2). The inhibition was a non-competitive manner and depended on the time for pre-incubation of ANS and MMP-7. At pH 4.5-9.5, the fluorescence of ANS was not changed by the addition of MMP-7. At pH 3.5, MMP-7 lacked activity, and the fluorescence of ANS was increased by the addition of MMP-7. These results suggest that at pH 4.5-9.5, the sulphonic group of ANS binds with MMP-7 through electrostatic interaction, whereas at pH 3.5, the anilinonaphthalene group of ANS binds with MMP-7 through hydrophobic interaction.