Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

On the Stabilizing Effect of Aspartate and Glutamate and Its Counteraction by Common Denaturants.

By performing differential scanning calorimetry(DSC) measurements on RNase A, we studied the stabilization provided by the addition of potassium aspartate(KAsp) or potassium glutamate (KGlu) and found that it leads to a significant increase in the denaturation temperature of the protein. The stabilization proves to be mainly entropic in origin. A counteraction of the stabilization provided by KAsp or KGlu is obtained by adding common denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate. A rationalization of the experimental data is devised on the basis of a theoretical approach developed by one of the authors. The main contribution to the conformational stability of globular proteins comes from the gain in translational entropy of water and co-solute ions and/or molecules for the decrease in solvent-excluded volume associated with polypeptide folding (i.e., there is a large decrease in solvent-accessible surface area). The magnitude of this entropic contribution increases with the number density and volume packing density of the solution. The two destabilizing contributions come from the conformational entropy of the chain, which should not depend significantly on the presence of co-solutes, and from the direct energetic interactions between co-solutes and the protein surface in both the native and denatured states. It is the magnitude of the latter that discriminates between stabilizing and destabilizing agents.

The anticancer peptide LL-III alters the physico-chemical properties of a model tumor membrane promoting lipid bilayer permeabilization.

LL-III is an anticancer peptide and has the ability to translocate across tumor cell membranes, which indicates that its action mechanism could be non-membranolytic. However, the exact mechanism through which the peptide gains access into the cell cytoplasm is still unknown. Here, we use a plethora of physico-chemical techniques to characterize the interaction of LL-III with liposomes mimicking the lipid matrix of the tumor cell membrane and its effect on the microstructure and thermotropic properties of the membrane. Furthermore, the effect of the presence of Ca2+ cations at physiological concentration was also investigated. For comparison, the interaction of LL-III with liposomes mimicking the normal cell membrane was also studied. Our results show that the peptide selectively interacts with the model tumor cell membrane. This interaction does not disrupt the lipid bilayer but deeply alters its properties by promoting lipid lateral reorganization and increasing membrane permeability. Overall, our data provide a molecular level description of the interaction of the peptide with the model tumor membrane and are fully consistent with the non-membranolytic action mechanism.

Investigating the Interaction of an Anticancer Nucleolipidic Ru(III) Complex with Human Serum Proteins: A Spectroscopic Study.

Ruthenium(III) complexes are very promising candidates as metal-based anticancer drugs, and several studies have supported the likely role of human serum proteins in the transport and selective delivery of Ru(III)-based compounds to tumor cells. Herein, the anticancer nanosystem composed of an amphiphilic nucleolipid incorporating a Ru(III) complex, which we named DoHuRu, embedded into the biocompatible cationic lipid DOTAP, was investigated as to its interaction with two human serum proteins thought to be involved in the mechanism of action of Ru(III)-based anticancer drugs, i.e., human serum albumin (HSA) and human transferrin (hTf). This nanosystem was studied in comparison with the simple Ru(III) complex named AziRu, a low molecular weight metal complex previously designed as an analogue of NAMI-A, decorated with the same ruthenium ligands as DoHuRu but devoid of the nucleolipid scaffold and not inserted in liposomal formulations. For this study, different spectroscopic techniques, i.e., Fluorescence Spectroscopy and Circular Dichroism (CD), were exploited, showing that DoHuRu/DOTAP liposomes can interact with both serum proteins without affecting their secondary structures.

A De Novo-Designed Type 3 Copper Protein Tunes Catechol Substrate Recognition and Reactivity.

De novo metalloprotein design is a remarkable approach to shape protein scaffolds toward specific functions. Here, we report the design and characterization of Due Rame 1 (DR1), a de novo designed protein housing a di-copper site and mimicking the Type 3 (T3) copper-containing polyphenol oxidases (PPOs). To achieve this goal, we hierarchically designed the first and the second di-metal coordination spheres to engineer the di-copper site into a simple four-helix bundle scaffold. Spectroscopic, thermodynamic, and functional characterization revealed that DR1 recapitulates the T3 copper site, supporting different copper redox states, and being active in the O2 -dependent oxidation of catechols to o-quinones. Careful design of the residues lining the substrate access site endows DR1 with substrate recognition, as revealed by Hammet analysis and computational studies on substituted catechols. This study represents a premier example in the construction of a functional T3 copper site into a designed four-helix bundle protein.

The C-terminus of the GKY20 antimicrobial peptide, derived from human thrombin, plays a key role in its membrane perturbation capability.

Previously, we characterized in detail the mechanism of action of the antimicrobial peptide GKY20, showing that it selectively perturbs the bacterial-like membrane employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. Here, we used a combination of biophysical techniques to similarly characterize the antimicrobial activity as well as the membrane perturbing capability of GKY10, a much shorter version of the GKY20 peptide. GKY10 is only half of the parent peptide and consists of the last 10 amino acids (starting from the C-terminus) of the full-length peptide. Despite a large difference in length, we found that GKY10, like the parent peptide, retains the ability to adopt a helical structure and to induce lipid segregation upon membrane binding. Overall, our results suggest that the amino acid sequence of GKY10 is responsible for most of the observed behaviors of GKY20. Our results shed further light on the mechanism of action of the full-length peptide and provide useful information for the design and development of new peptides that serve as antimicrobial agents.

Binding Properties of RNA Quadruplex of SARS-CoV-2 to Berberine Compared to Telomeric DNA Quadruplex.

Previous studies suggest that berberine, an isoquinoline alkaloid, has antiviral potential and is a possible therapeutic candidate against SARS-CoV-2. The molecular underpinnings of its action are still unknown. Potential targets include quadruplexes (G4Q) in the viral genome as they play a key role in modulating the biological activity of viruses. While several DNA-G4Q structures and their binding properties have been elucidated, RNA-G4Qs such as RG-1 of the N-gene of SARS-CoV-2 are less explored. Using biophysical techniques, the berberine binding thermodynamics and the associated conformational and hydration changes of RG-1 could be characterized and compared with human telomeric DNA-G4Q 22AG. Berberine can interact with both quadruplexes. Substantial changes were observed in the interaction of berberine with 22AG and RG-1, which adopt different topologies that can also change upon ligand binding. The strength of interaction and the thermodynamic signatures were found to dependent not only on the initial conformation of the quadruplex, but also on the type of salt present in solution. Since berberine has shown promise as a G-quadruplex stabilizer that can modulate viral gene expression, this study may also contribute to the development of optimized ligands that can discriminate between binding to DNA and RNA G-quadruplexes.

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III.

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.

Environment-Sensitive Fluorescent Labelling of Peptides by Luciferin Analogues.

Environment-sensitive fluorophores are very valuable tools in the study of molecular and cellular processes. When used to label proteins and peptides, they allow for the monitoring of even small variations in the local microenvironment, thus acting as reporters of conformational variations and binding events. Luciferin and aminoluciferin, well known substrates of firefly luciferase, are environment-sensitive fluorophores with unusual and still-unexploited properties. Both fluorophores show strong solvatochromism. Moreover, luciferin fluorescence is influenced by pH and water abundance. These features allow to detect local variations of pH, solvent polarity and local water concentration, even when they occur simultaneously, by analyzing excitation and emission spectra. Here, we describe the characterization of (amino)luciferin-labeled derivatives of four bioactive peptides: the antimicrobial peptides GKY20 and ApoBL, the antitumor peptide p53pAnt and the integrin-binding peptide RGD. The two probes allowed for the study of the interaction of the peptides with model membranes, SDS micelles, lipopolysaccharide micelles and Escherichia coli cells. Kd values and binding stoichiometries for lipopolysaccharide were also determined. Aminoluciferin also proved to be very well-suited to confocal laser scanning microscopy. Overall, the characterization of the labeled peptides demonstrates that luciferin and aminoluciferin are previously neglected environment-sensitive labels with widespread potential applications in the study of proteins and peptides.

Similarities and differences for membranotropic action of three unnatural antimicrobial peptides.

Previously, we described the design and synthesis of three nine-residue AMPs, P9Nal(SS), P9Trp(SS), and P9Nal(SR), showing high stability in serum and broad spectrum antimicrobial activity. The peptides P9Trp(SS) and P9Nal(SR) differ from P9Nal(SS) for the replacement of the two 2Nal residues with Trp residues and for the replacement of the two Cys (StBu) with Cys (tBu) residues, respectively. These changes led to peptides with a lower hydrophobicity respect to the P9Nal(SS). Interestingly, the three peptides have very similar activity against Gram-negative bacteria. Instead, they exhibit a significant difference towards Gram-positive bacteria, being P9Nal(SS) the most active. In order to evaluate the impact of amino acids substitution on membranotropic activity and rationalize the observed effects in vivo, here, we report the detailed biophysical characterization of the interaction between P9Nal(SR) and P9Trp(SS) and liposomes by combining differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The comparison with the results for the previously characterized P9Nal(SS) peptide reveals similarities and differences on the interaction process and perturbation activities. It was found that the three peptides can penetrate at different extent inside the bilayer upon changing their conformation and inducing lipid domains formation, revealing that the formation of lipid domains is fundamental for the activity against Gram-negative bacteria. On the contrary, the dissimilar activity against Gram-positive bacteria well correlate with the different affinity of peptides for the lipoteichoic acid, a component selectively present in the cell wall of Gram-positive bacteria.

Molecular Dissection of dH3w, A Fluorescent Peptidyl Sensor for Zinc and Mercury.

Previously, we reported that fluorescent peptide dansyl-HPHGHW-NH2 (dH3w), designed on the repeats of the human histidine-rich glycoprotein, shows a turn-on response to Zn(II) and a complex response to Hg(II) characterized by a turn-off phase at low Hg(II) concentrations and a turn-on phase at high concentrations. As Hg(II) easily displaces Zn(II), dH3w is a useful probe for the environmental monitoring of Hg(II). In order to investigate the molecular basis of the metal selectivity and fluorescence response, we characterized three variants, dH3w(H1A), dH3w(H3A), and dH3w(H5A), in which each of the three histidine residues was changed to alanine, and two variants with a single fluorescent moiety, namely dH3w(W6A), in which the tryptophan residue at the C-terminus was changed to alanine, and AcH3w, in which the N-terminal dansyl moiety was substituted by an acetyl group. These variants allowed us to demonstrate that all the histidine residues are essential for a strong interaction with Zn(II), whereas two histidine residues (in particular His5) and the dansyl group are necessary to bind Hg(II). The data reported herein shed light on the molecular behavior of dH3w, thus paving the way to the rational designing of further and more efficient fluorescent peptidyl probes for Hg(II).

A signalling cascade involving receptor-activated phospholipase A2, glycerophosphoinositol 4-phosphate, Shp1 and Src in the activation of cell motility.

BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.

Membrane disintegration by the antimicrobial peptide (P)GKY20: lipid segregation and domain formation.

Antimicrobial peptides (AMPs) are membrane-active peptides with a broad spectrum of activity against different pathogenic organisms and they represent promising new drugs to overcome the emergence of resistance to antibiotics in bacteria. (P)GKY20 is an antimicrobial peptide with a low hemolytic effect on eukaryotic cells and a strong antimicrobial activity especially against Gram-negative bacteria. However, its mechanism of action is still unknown. Here, we use fluorescence spectroscopy and differential scanning calorimetry combined with atomic force microscopy to characterise the binding of (P)GKY20 with model biomembranes and its effect on the membrane's microstructure and thermotropic properties. We found that (P)GKY20 selectively perturbs the bacterial-like membrane via a carpet-like mechanism employing peptide conformational changes, lipid segregation and domain formation as key steps in promoting membrane disruption. These results shed a first light on the action mechanism of (P)GKY20 and could represent an important contribution to the development of new peptides serving as antimicrobial agents.

Counteraction ability of TMAO toward different denaturing agents.

Differential scanning calorimetry measurements performed on RNase A in aqueous binary solutions containing different concentrations of urea, tetramethylurea, guanidinium chloride, and guanidinium thiocyanate, and in aqueous ternary solutions, containing the same denaturants plus 1 M trimethylamine N-oxide, TMAO, demonstrate that the latter has a general counteracting ability at pH 7.0, but not at pH 4.0. Experimental data rule out the idea that counteraction originates from direct interactions between TMAO molecules and denaturing agents. A rationalization is provided on the basis of a theoretical approach grounded on the solvent-excluded volume effect, whose magnitude depends on the density of aqueous solutions.

Counteraction of denaturant-induced protein unfolding is a general property of stabilizing agents.

DSC measurements on RNase A at neutral pH show that five stabilizing agents, namely trimethylamine N-oxide, glucose, sucrose, betaine and sodium sulfate, can counteract the destabilizing action of urea, sodium perchlorate, guanidinium chloride and guanidinium thiocyanate. This is an important finding inferring that counteraction has a common physical origin, regardless of the chemical differences among the stabilizing agents and among the destabilizing ones. A rationalization is provided grounded on the following line of reasoning: (a) the decrease in solvent-excluded volume effect is the main stabilizing contribution of the native state; (b) its magnitude increases on increasing the density of the aqueous solution; (c) the density increases significantly in the ternary solutions containing water, a stabilizing agent and a destabilizing one, as indicated by the present experimental data.

DMSO-induced perturbation of thermotropic properties of cholesterol-containing DPPC liposomes.

Dimethyl sulfoxide (DMSO) is a universal solvent widely used in many fields, from basic research to industrial applications. At low concentration, it is the most important cryoprotectant agent against cellular damage caused during a freeze-thaw cycle. Although the effects of this cosolvent on the physico-chemical properties of a lipid bilayer have been extensively studied with both in vitro and in vivo experiments, the molecular mechanism of cryopreservation is not completely understood. Cholesterol (Chol) represents one of the essential cell membrane component and is fundamental to maintain the integrity and fluidity of the membrane. Here we report a study on the effect of DMSO on the stability of Chol-containing model membranes. We investigated the effect of DMSO on thermal stability of model membranes formed by dipalmitoylphospatidylcholine (DPPC) and DPPC/Chol by means of Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC) measurements. It is well known that cholesterol reduces the thermal stability of DPPC vesicles and also the pre-transition is abolished. Our results show that DMSO induces a stabilization of the lipid bilayer of DPPC liposomes increasing both the pre- and main transition temperatures. In DPPC/Chol liposomes a similar thermal stabilization was observed for the main transition indicating that DMSO is capable to stabilize the lipid bilayer even in the presence of the sterol. Moreover, by direct inspection of the hydration degree of the lipid bilayers, we evidenced the role played by DMSO on the thermal stability of the membrane as connected to the hydration of the polar head groups.

Proline 235 plays a key role in the regulation of the oligomeric states of Thermotoga maritima Arginine Binding Protein.

The Arginine Binding Protein isolated from Thermotoga maritima (TmArgBP) is a protein endowed with several peculiar properties. We have previously shown that TmArgBP dimerization is a consequence of the swapping of the C-terminal helix. Here we explored the structural determinants of TmArgBP domain swapping and oligomerization. In particular, we report a mutational analysis of the residue Pro235, which is located in the hinge region of the swapping dimer. This residue was either replaced with a Gly-Lys dipeptide (TmArgBP(P235GK)) or a Gly residue (TmArgBP(P235G)). Different forms of these mutants were generated and extensively characterized using biophysical techniques. For both TmArgBP(P235GK) and TmArgBP(P235G) mutants, the occurrence of multiple oligomerization states (monomers, dimers and trimers) was detected. The formation of well-folded monomeric forms for these mutants indicates that the dimerization through C-terminal domain swapping observed in wild-type TmArgBP is driven by conformational restraints imposed by the presence of Pro235 in the hinge region. Molecular dynamics studies corroborate this observation by showing that Gly235 assumes conformational states forbidden for Pro residues in the TmArgBP(P235G) monomer. Unexpectedly, the trimeric forms present: (a) peculiar circular dichroism spectra, (b) a great susceptibility to heating, and (c) the ability to bind the Thioflavin T dye. The present findings clearly demonstrate that single-point mutations have an important impact on the TmArgBP oligomerization process. In a wider context, they also indicate that proteins endowed with an intrinsic propensity to swap have an easy access to states with altered structural and, possibly, functional properties.

On the microscopic and mesoscopic perturbations of lipid bilayers upon interaction with the MPER domain of the HIV glycoprotein gp41.

The effect of the 665-683 fragment of the HIV fusion glycoprotein 41, corresponding to the MPER domain of the protein and named gp41MPER, on the microscopic structure and mesoscopic arrangement of palmitoyl oleoyl phosphatidylcholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (CHOL) lipid bilayers is analyzed. The microscopic structuring of the bilayers has been studied by Electron Spin Resonance (ESR) spectroscopy, using glycerophosphocholines spin-labelled in different positions along the acyl chain. Transitions of the bilayer liquid crystalline state have been also monitored by Differential Scanning Calorimetry (DSC). Changes of the bilayers morphology have been studied by determining the dimension of the liposomes through Dynamic Light Scattering (DLS) measurements. The results converge in showing that the sample preparation procedure, the bilayer composition and the peptide/lipid ratio critically tune the lipid response to the peptide/membrane interaction. When gp41MPER is added to preformed liposomes, it positions at the bilayer interface and the lipid perturbation is limited to the more external segments. In contrast, if the peptide is mixed with the lipids during the liposome preparation, it assumes a trans-membrane topology. This happens at all peptide/lipid ratios for fluid POPC bilayers, while in the case of rigid POPC/SM/CHOL membranes a minimum ratio has to be reached, thus suggesting peptide self-aggregation to occur. Peptide insertion results in a dramatic increase of the lipid ordering and bilayer stiffening, which reflect in significant changes in liposome average dimension and distribution. The biological implications of these findings are discussed.

Binding of a type 1 RIP and of its chimeric variant to phospholipid bilayers: evidence for a link between cytotoxicity and protein/membrane interactions.

Ribosome-inactivating proteins (RIPs) are enzymes, almost all identified in plants, able to kill cells by depurination of rRNAs. Recently, in order to improve resistance to proteolysis of a type 1 RIP (PD-L4), we produced a recombinant chimera combining it with a wheat protease inhibitor (WSCI). Resulting chimeric construct, named PD-L4UWSCI, in addition to present the functions of the two domains, shows also an enhanced cytotoxic action on murine cancer cells when compared to PD-L4. Since different ways of interaction of proteins with membranes imply different resulting effects on cells, in this study we investigate conformational stability of PD-L4 and PD-L4UWSCI and their interaction with membrane models (liposomes). Circular dichroism analysis and differential scanning calorimetry measurements indicate that PD-L4 and PD-L4UWSCI present high and similar conformational stability, whereas analysis of their binding to liposomes, obtained by isothermal titration calorimetry and differential scanning calorimetry, clearly indicate that chimera is able to interact with biomembranes more effectively. Overall, our data point out that WSCI domain, probably because of its flexibility in solution, enhances the chimeric protein interaction with membrane lipid surfaces without however destabilizing the overall protein structure. Analysis of interactions between RIPs or RIP based conjugates and lipid surfaces could provide novel insights in the search of more effective selective membrane therapeutics.

Differential thermodynamic behaviours of the extra-cellular regions of two Ser/Thr PrkC kinases revealed by calorimetric studies.

Eukaryotic-type Ser/Thr protein-kinases are critical mediators of developmental changes and host pathogen interactions in bacteria. Although with lower abundance compared to their homologues from eukaryotes, Ser/Thr protein-kinases (STPK) are widespread in gram positive bacteria, where they regulate several cellular functions. STPKs belong to the protein kinase family named as one-component signal transduction systems, which combine both sensing and regulating properties. Thermodynamic investigations of sensing extra-cellular portions of two important Ser-Thr kinases, PrkC, from Staphylococcus aureus and Bacillus subtilis were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements, coupled with modelling studies. The study of thermodynamic properties of the two domains is challenging since they share a modular domain organization. Consistently, DSC and CD data show that they present similar thermodynamic behaviours and that folding/unfolding transitions do not fit a two-state folding model. However, the thermal unfolding of the two proteins is differentially sensitive to pH. In particular, their unfolding is characteristic of modular structures at the neutral pH, with independent contributions of individual domains to folding. Differently, a cooperative unfolding is evidenced at acidic pH for the B. subtilis member, suggesting that a significant interaction between domains becomes valuable.