The Structural Stability of Membrane Proteins Revisited: Combined Thermodynamic and Spectral Phasor Analysis of SDS-induced Denaturation of a Thermophilic Cu(I)-transport ATPase.

Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.

Effects of Single and Double Mutants in Human Glucose-6-Phosphate Dehydrogenase Variants Present in the Mexican Population: Biochemical and Structural Analysis.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent human enzymopathy, affecting over 400 million people globally. Worldwide, 217 mutations have been reported at the genetic level, and only 19 have been found in Mexico. The objective of this work was to contribute to the knowledge of the function and structure of three single natural variants (G6PD A+, G6PD San Luis Potosi, and G6PD Guadalajara) and a double mutant (G6PD Mount Sinai), each localized in a different region of the three-dimensional (3D) structure. In the functional characterization of the mutants, we observed a decrease in specific activity, protein expression and purification, catalytic efficiency, and substrate affinity in comparison with wild-type (WT) G6PD. Moreover, the analysis of the effect of all mutations on the structural stability showed that its presence increases denaturation and lability with temperature and it is more sensible to trypsin digestion protease and guanidine hydrochloride compared with WT G6PD. This could be explained by accelerated degradation of the variant enzymes due to reduced stability of the protein, as is shown in patients with G6PD deficiency.

ANS Interacts with the Ca2+-ATPase Nucleotide Binding Site.

The binding of 8-anilino-1-naphthalene sulfonate (ANS) to the nucleotide binding domain (N-domain) of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) was studied. Molecular docking predicted two ANS binding modes (BMI and BMII) in the nucleotide binding site. The molecular interaction was confirmed as the fluorescence intensity of ANS was dramatically increased when in the presence of an engineered recombinant N-domain. Molecular dynamics simulation showed BMI (which occupies the ATP binding site) as the mode that is stable in solution. The above was confirmed by the absence of ANS fluorescence in the presence of a fluorescein isothiocyanate (FITC)-labeled N-domain. Further, the labeling of the N-domain with FITC was hindered by the presence of ANS, i.e., ANS was bound to the ATP binding site. Importantly, ANS displayed a higher affinity than ATP. In addition, ANS binding led to quenching the N-domain intrinsic fluorescence displaying a FRET pattern, which suggested the existence of a Trp-ANS FRET couple. Nonetheless, the chemical modification of the sole Trp residue with N-bromosuccinimide (NBS) discarded the existence of FRET and instead indicated structural rearrangements in the nucleotide binding site during ANS binding. Finally, Ca2+-ATPase kinetics in the presence of ANS showed a partial mixed-type inhibition. The Dixon plot showed the ANS-Ca2+-ATPase complex as catalytically active, hence supporting the existence of a functional dimeric Ca2+-ATPase in sarcoplasmic reticulum vesicles. ANS may be used as a molecular platform for the development of more effective inhibitors of Ca2+-ATPase and appears to be a new fluorescent probe for the nucleotide binding site. Graphical Abstract Molecular docking of ANS to the nucleotide binding site of Ca2+-ATPase. ANS fluorescence increase reveals molecular interaction.

The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability.

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0 ±â€¯7.9 µM) and the K0.5 values determined for G6P (57.9 ±â€¯2.5 and 104.5 ±â€¯9.3 µM in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.

Formation of large oligomers of DOPAL-modified α-synuclein is modulated by the oxidation of methionine residues located at C-terminal domain.

The formation of neurotoxic oligomers of the presynaptic protein α-Synuclein (aSyn) is suggested to be associated with Parkinson's disease neurodegeneration. In this respect, it was demonstrated that the aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), a product from the enzymatic oxidation of dopamine, is capable of stabilizing potentially toxic aSyn oligomers via formation of covalent adducts with Lys residues of the protein. In addition, DOPAL-induced production of reactive oxygen species (ROS) leads to the oxidation of aSyn's Met residues to Met-sulfoxide. Recently, our group pointed out that the pre-oxidation of all-four Met residues of aSyn, upon treatment with H2O2, decreases the formation of large aSyn-DOPAL oligomers, which are suggested to be more toxic to neurons than the corresponding small oligomers (Carmo-Gonçalves et al., Biochem. Biophys. Res. Comm. 505, 295-301. 2018). By using a series of Met to Val mutants of aSyn, we demonstrated that the ability of aSyn to scavenge ROS/H2O2 generated from DOPAL oxidation is primarily dependent on Met residues located at the C-terminal domain of the protein, which contrasts with the reactivity of aSyn against H2O2 itself in which N-terminal Met residues (notably Met5) were more readily oxidized. Interestingly, the substitution of C-terminal Met residues (particularly Met127) by Val increased the formation of DOPAL-induced large oligomers in comparison with the wild-type protein. In this context, we demonstrated that the hydrophobicity of aSyn monomer, which is affected distinctively by the oxidation of N- versus C-terminal methionines, is correlated with the formation of large (but not small) oligomers of aSyn mediated by DOPAL.

How the presence of a small molecule affects the complex coacervation between lactoferrin and ß-lactoglobulin.

Heteroprotein complex coacervation corresponds to the formation of two liquid phases in equilibrium induced by the interaction of two oppositely charged proteins. The more concentrated phase known as coacervate phase, has attracted interest from several fields of science due to its potential applications for example for encapsulation and delivery of bioactives. Prior such application, it is necessary to understand how the presence of small ligands affects the complex coacervation. In this work, we report on the interaction of small ligand with individual proteins ß-lactoglobulin (ß-LG) and lactoferrin (LF) and consequences on their complex coacervation. ANS (8-Anilinonaphthalene-1-sulfonic acid), a fluorescent probe, was used as model ligand. While ANS did not interact with ß-LG, it presented two sets of binding sites with LF inducing its self-aggregation. Depending on its concentration, ANS modulated the shape of ß-LG-LF macromolecular assembly. Coacervates were observed for ANS/LF molar ratio <25 against amorphous aggregates for higher ANS/LF molar ratios. A maximum loading capacity of around 40mg of ANS per gram of LF in the formed heteroprotein coacervates was reached.

Opposing effects of Na+ and K+ on the thermal stability of Na+,K(+)-ATPase.

Folding and structural stability are key factors for the proper biological function of proteins. Na(+),K(+)-ATPase is an integral membrane protein involved in the active transport of Na(+) and K(+) across the plasma membrane. In this work we characterized the effects of K(+) and Na(+) on the thermal inactivation of Na(+),K(+)-ATPase, evaluating both catalytic and transport capacities of the pump. Both activities of the enzyme decrease with the preincubation time as first-order kinetics. The thermal inactivation of Na(+),K(+)-ATPase is simultaneous with a conformational change detected by tryptophan and 1-aniline-8-naphtalenesulfonate (ANS) fluorescence. The kinetic coefficient of thermal inactivation was affected by the presence of Na(+) and K(+) (or Rb(+)) and the temperature of the preincuabtion media. Our results show that K(+) or Rb(+) stabilize the enzyme, while Na(+) decreases the stability of Na(+),K(+)-ATPase. Both effects are exerted by the specific binding of these cations to the pump. Also, we provided strong evidence that the Rb(+) (or K(+)) stabilization effect is due to the occlusion of these cations into the enzyme. Here, we proposed a minimal kinetic model that explains the behavior observed in the experimental results and allows a better understanding of the results presented by other researchers. The thermal inactivation process was also analyzed according to Kramer's theory.

Identification of a novel ligand binding motif in the transthyretin channel.

The design of therapeutic compounds targeting transthyretin (TTR) is challenging due to the low specificity of interaction in the hormone binding site. Such feature is highlighted by the interactions of TTR with diclofenac, a compound with high affinity for TTR, in two dissimilar modes, as evidenced by crystal structure of the complex. We report here structural analysis of the interactions of TTR with two small molecules, 1-amino-5-naphthalene sulfonate (1,5-AmNS) and 1-anilino-8-naphthalene sulfonate (1,8-ANS). Crystal structure of TTR:1,8-ANS complex reveals a peculiar interaction, through the stacking of the naphthalene ring between the side-chain of Lys15 and Leu17. The sulfonate moiety provides additional interaction with Lys15' and a water-mediated hydrogen bond with Thr119'. The uniqueness of this mode of ligand recognition is corroborated by the crystal structure of TTR in complex with the weak analogue 1,5-AmNS, the binding of which is driven mainly by hydrophobic partition and one electrostatic interaction between the sulfonate group and the Lys15. The ligand binding motif unraveled by 1,8-ANS may open new possibilities to treat TTR amyloid diseases by the elucidation of novel candidates for a more specific pharmacophoric pattern.

Kinetics and thermodynamics of the interaction of 1-anilino-naphthalene-8-sulfonate with proteins.

Although 1-anilino-naphthalene-8-sulfonate (ANS) has been widely used in protein folding and binding studies, the detailed mechanism of this interaction is not fully understood. In this work the binding of ANS was analyzed at pre-equilibrium and equilibrium conditions using bovine serum albumin (BSA) as model. We employed a combined approach including the analysis of fluorescence, near-UV circular dichroism and isothermal titration calorimetric data. Experiments at equilibrium with these techniques identify three ANS molecules bound at hydrophobic cavities in BSA. Pre-equilibrium fluorescence analysis unambiguously indicated that the binding of ANS at hydrophobic cavities of BSA occurs at two different and independent classes of sites with similar affinities and quantum yields, two features that are undetectable by the equilibrium analysis. The binding of ANS to the first site is thermodynamically favored by similar contributions of the enthalpic (DeltaH = -22 kJ/mol) and entropic terms (-TDeltaS = -17 kJ/mol), while the binding to the second site is enthalpically driven (DeltaH = -31 kJ/mol; -TDeltaS = -0.6 kJ/mol). Complementary information from molecular docking showed three ANS molecules bound at hydrophobic cavities in BSA subdomains IIA and IIIA with binding affinities in the order of those found experimentally and three additional ANS molecules bound at water exposed sites.

Cognate DNA stabilizes the tumor suppressor p53 and prevents misfolding and aggregation.

The tumor suppressor protein p53 is a nuclear protein that serves as an important transcription factor. The region responsible for sequence-specific DNA interaction is located in its core domain (p53C). Although full-length p53 binds to DNA as a tetramer, p53C binds as a monomer since it lacks the oligomerization domain. It has been previously demonstrated that two core domains have a dimerization interface and undergo conformational change when bound to DNA. Here we demonstrate that the interaction with a consensus DNA sequence provides the core domain of p53 with enhanced conformational stability at physiological salt concentrations (0.15 M). This stability could be either increased or abolished at low (0.01 M) or high (0.3 M) salt concentrations, respectively. In addition, interaction with the cognate sequence prevents aggregation of p53C into an amyloid-like structure, whereas binding to a nonconsensus DNA sequence has no effect on p53C stability, even at low ionic strength. Strikingly, sequence-specific DNA binding also resulted in a large stabilization of full-length p53, whereas nonspecific sequence binding led to no stabilization. The effects of cognate DNA could be mimicked by high concentrations of osmolytes such as glycerol, which implies that the stabilization is caused by the exclusion of water. Taken together, our results show an enhancement in protein stability driven by specific DNA recognition. When cognate DNA was added to misfolded protein obtained after a pressurization cycle, the original conformation was mostly recovered. Our results may aid the development of therapeutic approaches to prevent misfolded species of p53.

Biophysical characterisation and urea-induced unfolding of recombinant Yarrowia lipolytica sterol carrier protein-2.

We report a biophysical characterisation of apo-sterol carrier protein-2 from Yarrowia lipolytica (YLSCP-2) and its urea-induced unfolding followed by intrinsic tryptophan fluorescence, far-UV CD, ANS binding, and small angle X-ray scattering (SAXS). The unfolding is described as a three-step process. The first steps, between 1 and 2 M urea, have well-defined cooperative character and are related to the break down of most of the tertiary and secondary structure. The third step, at higher urea concentrations, is characterised by the disruption of residual interactions involving the single tryptophan. A 3D structure model for the YLSCP2 monomer was built by homology, which account for the fluorescence and CD spectroscopy data and is consistent with the binding mode observed for other SCP2. SAXS and cross-linking experiments suggest that YLSCP2 dimerise at approximately 70 microM concentration.

Unraveling multistate unfolding of pig kidney fructose-1,6-bisphosphatase using single tryptophan mutants.

Pig kidney fructose-1,6-bisphosphatase is a homotetrameric enzyme which does not contain tryptophan. In a previous report the guanidine hydrochloride-induced unfolding of the enzyme has been described as a multistate process [Reyes, A. M., Ludwig, H. C., Yañez, A. J., Rodriguez, P. H and Slebe, J. C. (2003) Biochemistry 42, 6956-6964]. To monitor spectroscopically the unfolding transitions, four mutants were constructed containing a single tryptophan residue either near the C1-C2 or the C1-C4 intersubunit interface of the tetramer. The mutants were shown to retain essentially all of the structural and kinetic properties of the enzyme isolated from pig kidney. The enzymatic activity, intrinsic fluorescence, size-exclusion chromatographic profiles and 1-anilinonaphthalene-8-sulfonate binding by the mutants were studied under unfolding equilibrium conditions. The unfolding profiles were multisteps, and formation of hydrophobic structures was detected. The enzymatic activity of wild-type and mutant FBPases as a function of guanidine hydrochloride concentration showed an initial enhancement (maximum approximately 30%) followed by a biphasic decay. The activity and fluorescence results indicate that these transitions involve conformational changes in the fructose-1,6-bisphosphate and AMP domains. The representation of intrinsic fluorescence data as a 'phase diagram' reveals the existence of five intermediates, including two catalytically active intermediates that have not been previously described, and provides the first spectroscopic evidence for the formation of dimers. The intrinsic fluorescence unfolding profiles indicate that the dimers are formed by selective disruption of the C1-C2 interface.

Thermal-unfolding reaction of triosephosphate isomerase from Trypanosoma cruzi.

Thermal denaturation of triosephosphate isomerase from Trypanosoma cruzi was studied by circular dicrhoism and fluorescence spectroscopies. The unfolding transition was found to be highly irreversible even at the very early stages of the reaction. Kinetic studies, allowed us to identify consecutive reactions. Firstly, only the tryptophan environment is altered. Next, changes on the secondary structure and hydrophobic surface exposure measured by 1-anilino-8-naphthalenesulfonate (ANS) binding were observed. Further conformational changes imply additional modifications on the secondary and tertiary structures and release of the hydrophobic dye leading to the formation of the unfolded state that is prone to aggregate.

Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH.

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability.

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.

Differential scanning calorimetry as a tool to estimate binding parameters in multiligand binding proteins.

The stability of proteins and their interactions with other molecules is a topic of special interest in biochemistry because many cellular processes depend on that. New methods and approaches are constantly developed to elucidate the energetics of biomolecular recognition. In this sense, the application of the theory of macromolecular unfolding linked to ligand binding to differential scanning calorimetry (DSC) has proved to be a useful tool to simultaneously characterize the energetics of unfolding and binding. Although the general theory is well known, the applicability of DSC to study the interaction of biomolecules is not common. In the current work, we estimated the binding parameters of 8-anilinonaphthalene-1-sulfonic acid to human serum albumin using DSC. This model system was chosen due to both the complex stoichiometry and the moderate binding constants. From DSC curves acquired at different ligand concentrations, we obtained the number of bound ligands, the binding constants, and the binding enthalpy for each independent binding site. Compared with those parameters determined by titration calorimetry, the results highlight the potentiality of DSC to estimate binding parameters in multiligand binding proteins.

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.

Ligand-induced thermostability in proteins: thermodynamic analysis of ANS-albumin interaction.

A comparative thermodynamic study of the interaction of anilinonaphthalene sulfonate (ANS) derivatives with bovine serum albumin (BSA) was performed by using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The chemically related ligands, 1,8-ANS and 2,6-ANS, present a similar affinity for BSA with different binding energetics. The analysis of the binding driving forces suggests that not only hydrophobic effect but also electrostatic interactions are relevant, even though they have been extensively used as probes for non-polar domains in proteins. Ligand association leads to an increase in protein thermostability, indicating that both dyes interact mainly with native BSA. ITC data show that 1,8-ANS and 2,6-ANS have a moderate affinity for BSA, with an association constant of around 1-9x10(5) M(-1) for the high-affinity site. Ligand binding is disfavoured by conformational entropy. The theoretical model used to simulate DSC data satisfactorily reproduces experimental thermograms, validating this approach as one which provides new insights into the interaction between one or more ligands with a protein. By comparison with 1,8-ANS, 2,6-ANS appears as a more "inert" probe to assess processes which involve conformational changes in proteins.

Folding and stability of a coiled-coil investigated using chemical and physical denaturing agents: comparative analysis of polymerized and non-polymerized forms of alpha-tropomyosin.

alpha-Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, which participates in the regulation of muscle contraction. Unlike Tm purified from vertebrate muscle, recombinant Tm expressed in Escherichia coli is not acetylated at the N-terminal residue and loses the capacity to undergo head-to-tail polymerization, to bind actin and to inhibit actomyosin ATPase activity. These functions are restored by fusion of an N-terminal Ala-Ser (AS) dipeptide tail to recombinant Tm. Here, we have employed chemical (guanidine hydrochloride and urea) and physical (elevated hydrostatic pressures and low temperatures) denaturing agents to compare the structural stabilities of polymeric alanine-serine-tropomyosin (ASTm, containing the AS dipeptide) and dimeric "non-fusion" Tm (nfTm, i.e., not containing the AS dipeptide). Binding of the hydrophobic fluorescent dye bis-ANS, circular dichroism and size-exclusion chromatography were used to monitor the stabilities and state of association of both proteins under different solution conditions. Bis-ANS binding was markedly decreased at low concentrations (<1M) of GdnHCl or urea, whereas the secondary structures of both ASTm and nfTm were essentially unaffected in the same range of denaturant concentrations. These results suggest local unfolding of bis-ANS binding domains prior to global unfolding of Tm. In contrast, increased bis-ANS binding was observed when Tm was submitted to high pressures or to low temperatures, implying increased exposure of hydrophobic domains in the protein. Taken together, the different sensitivities of ASTm and nfTm to different denaturing agents support the notion that, at close to physiological conditions, head-to-tail interactions in polymerized ASTm are predominantly stabilized by electrostatic interactions between adjacent Tm dimers, whereas non-polar interactions appear to play a major role in the stability of the coiled-coil structure of individual Tm dimers.

Urea-induced denaturation of beta-trypsin: an evidence for a molten globule state.

The denaturation of beta-trypsin induced by urea was investigated by fluorescence and circular dichroism. A transient denatured state was found at 2 M urea in both intrinsic fluorescence spectrum and bis-(8-anilino-1-naphtalene sulfonate) (bis-ANS) binding. In addition, the absence of tertiary contacts and presence of secondary structure for this state, are consistent with an intermediate equilibrium state having features of molten globule.