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Circular dichroism (CD) spectroscopy is a well-established biophysical technique used to investigate the structure of molecules. The analysis of a protein CD spectrum depends on the quality of the original CD data, which can be affected by the sample purity, background absorption of the additives/solvent/buffer, the choice of the parameters used for data collection, etc. In this paper, the CD spectrum of myoglobin was used as a model to exploit how variations on each data collection parameter could affect the final protein CD spectrum and, the subsequent effect of them on the quantitative analysis of protein secondary structure. Bioinformatics analysis carried out with SESCA package and PDBMD2CD server predicted a theoretical myoglobin CD spectrum, and a Monte Carlo-like model was implemented to estimate the uncertainty in secondary structure predictions performed with CDSSTR, Selcon 3 and ContinLL algorithms. An inappropriate choice of data collection parameters can lead to a misinterpretation of the CD data in terms of the protein structural content.

Antimicrobial peptides (AMPs) are important constituents of the innate immunity system of all living organisms. They participate in the first line of defense against invading pathogens such as viruses, bacteria, and fungi. In view of the increasing difficulties to treat infectious diseases due to the emergence of antibiotic-resistant bacterial strains, AMPs have great potential to control infectious diseases in humans and animals. In this study, two small peptides, RcAlb-PepI and RcAlb-PepII, were designed based on the primary structure of Rc-2S-Alb, a 2S albumin from the seed cake of Ricinus communis, and their antimicrobial activity assessed. RcAlb-PepII strongly inhibited the growth of Klebsiella pneumoniae and Candida parapsilosis, and induced morphological alterations in their cell surface. C. parapsilosis exposed to RcAlb-PepII presented higher cell membrane permeabilization and elevated content of reactive oxygen species. RcAlb-PepII also degraded and reduced the biofilm formation in C. parapsilosis and in K. pneumonia cells. Experimentally, RcAlb-PepII was not hemolytic and had low toxicity to mammalian cells. These are advantageous characteristics, which suggest that RcAlb-PepII is safe and apparently effective for its intended use and has great potential for the future development of an antimicrobial agent with the ability to kill or inhibit K. pneumoniae and C. parapsilosis cells.

Antimicrobial peptides are a large group of natural compounds which present promising properties for the pharmaceutical and food industries, such as broad-spectrum activity, potential for use as natural preservatives, and reduced propensity for development of bacterial resistance. Plantaricin 149 (Pln149), isolated from Lactobacillus plantarum NRIC 149, is an intrinsically disordered peptide with the ability to inhibit bacteria from the Listeria and Staphylococcus genera, and which is capable of promoting inhibition and disruption of yeast cells. In this study, the interactions of Pln149 with model membranes composed of zwitterionic and/or anionic phospholipids were investigated using a range of biophysical techniques, including isothermal titration calorimetry, surface tension measurements, synchrotron radiation circular dichroism spectroscopy, oriented circular dichroism spectroscopy, and optical microscopy, to elucidate these peptides’ mode of interactions and provide insight into their functional roles. In anionic model membranes, the binding of Pln149 to lipid bilayers is an endothermic process and induces a helical secondary structure in the peptide. The helices bind parallel to the surfaces of lipid bilayers and can promote vesicle disruption, depending on peptide concentration. Although Pln149 has relatively low affinity for zwitterionic liposomes, it is able to adsorb at their lipid interfaces, disturbing the lipid packing, assuming a similar parallel helix structure with a surface-bound orientation, and promoting an increase in the membrane surface area. Such findings can explain the intriguing inhibitory action of Pln149 in yeast cells whose cell membranes have a significant zwitterionic lipid composition.

Micro Exon Gene (MEG) proteins are thought to play major roles in the infection and survival of parasitic Schistosoma mansoni worms in host organisms. Here, the physical chemical properties of two small MEG proteins found in the genome of S. mansoni, named MEG-24 and MEG-27, were examined by a combination of biophysical techniques such as differential scanning calorimetry, tensiometry, circular dichroism, fluorescence, and electron spin resonance spectroscopies. The proteins are surface active and structurally arranged as cationic amphipathic α-helices that can associate with lipid membranes and cause their disruption. Upon adsorption to lipid membranes, MEG-27 strongly affects the fluidity of erythrocyte ghost membranes, whereas MEG-24 forms pores in erythrocytes without modifying the ghost membrane fluidity. Whole-mount in situ hybridization experiments indicates that MEG-27 and MEG-24 transcripts are located in the parasite esophagus and subtegumental cells, respectively, suggesting a relevant role of these proteins in the host-parasite interface. Taken together, these characteristics lead us to propose that these MEG proteins may interact with host cell membranes and potentially modulate the immune process using a similar mechanism as that described for α-helical membrane-active peptides.

Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.

The unordered secondary structural content of an intrinsically disordered protein (IDP) is susceptible to conformational changes induced by many different external factors, such as the presence of organic solvents, removal of water, changes in temperature, binding to partner molecules, and interaction with lipids and/or other ligands. In order to characterize the high-flexibility nature of an IDP, circular dichroism (CD) spectroscopy is a particularly useful method due to its capability of monitoring both subtle and remarkable changes in different environments, relative ease in obtaining measurements, the small amount of sample required, and the capability for sample recovery (sample not damaged) and others. Using synchrotron radiation as the light source for CD spectroscopy represents the state-of-the-art version of this technique with feasibility of accessing the lower wavelength UV region, and therefore presenting a series of advantages over conventional circular dichroism (cCD) to monitor a protein conformational behavior, check protein stability, detect ligand binding, and many others. In this paper, we have performed a comparative study using cCD and SRCD methods for investigating the secondary structure and the conformational behavior of natively unfolded proteins: MEG-14 and soybean trypsin inhibitor. We show that the SRCD technique greatly improves the analysis and accuracy of the studies on the conformations of IDPs.

BACKGROUND: The Micro-Exon Gene-14 (MEG-14) displays a remarkable structure that allows the generation of antigenic variation in Schistosomes. Previous studies showed that the soluble portion of the MEG-14 protein displays features of an intrinsically disordered protein and is expressed exclusively in the parasite esophageal gland. These features indicated a potential for interaction with host proteins present in the plasma and cells from ingested blood. METHODS: A yeast two-hybrid experiment using as bait the soluble domain of Schistosoma mansoni MEG-14 (sMEG-14) against a human leukocyte cDNA library was performed. Pull-down and surface plasmon resonance (SPR) experiments were used to validate the interaction between sMEG-14 and human S100A9. Synchrotron radiation circular dichroism (SRCD) were used to detect structural changes upon interaction between sMEG-14 and human S100A9. Feeding of live parasites with S100A9 attached to a fluorophore allowed the tracking of the fate of this protein in the parasite digestive system. RESULTS: S100A9 interacted with sMEG-14 consistently in yeast two-hybrid assay, pull-down and SPR experiments. SRCD suggested that MEG-14 acquired a more regular structure as a result of the interaction with S100A9. Accumulation of recombinant S100A9 in the parasite's esophageal gland, when ingested by live worms suggests that such interaction may occur in vivo. CONCLUSION: S100A9, a protein previously described to be involved in modulation of inflammatory response, was found to interact with sMEG-14. GENERAL SIGINFICANCE: Our results allow proposing a mechanism involving MEG-14 for the parasite to block inflammatory signaling, which would occur upon release of S100A9 when ingested blood cells are lysed.

Background Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria. Methods The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined. Results Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold. Conclusions The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method. General significance We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors., Highlights • Novel method for producing natively folded disulfide linked toxins • Co-expression in periplasmic space with chaperones • Crystal structure has the same structure/disulfide links as toxin from natural source. • Synchrotron radiation circular dichroism spectroscopy shows thermal stability. • Potential uses in studies of channel and receptor structure/function relationships

The micro-exon genes (MEG) of Schistosoma mansoni, a parasite responsible for the second most widely spread tropical disease, code for small secreted proteins with sequences unique to the Schistosoma genera. Bioinformatics analyses suggest the soluble domain of the MEG-14 protein will be largely disordered, and using synchrotron radiation circular dichroism spectroscopy, its secondary structure was shown to be essentially completely unfolded in aqueous solution. It does, however, show a strong propensity to fold into more ordered structures under a wide range of conditions. Partial folding was produced by increasing temperature (in a reversible process), contrary to the behavior of most soluble proteins. Furthermore, significant folding was observed in the presence of negatively charged lipids and detergents, but not in zwitterionic or neutral lipids or detergents. Absorption onto a surface followed by dehydration stimulated it to fold into a helical structure, as it did when the aqueous solution was replaced by nonaqueous solvents. Hydration of the dehydrated folded protein was accompanied by complete unfolding. These results support the identification of MEG-14 as a classic intrinsically disordered protein, and open the possibility of its interaction/folding with different partners and factors being related to multifunctional roles and states within the host.

Recent results from our laboratory have previously shown the purification of a small serine proteinase inhibitor (PI), named CaTI1, from Capsicum annuum seeds. This work demonstrated the characterization of CaTI now named CaTI1, and the identification of two other small serine PIs, named CaTI2 and CaTI3, also present in these seeds. CaTI1 presented molecular mass of 6 kDa and pI value of ∼9.0. CaTI1 inhibited both trypsin and chymotrypsin with inhibition constants (Ki and Ki') of 14 and 2.8 nM for trypsin and 4.3 and 0.58 nM for chymotrypsin, respectively. Circular dichroism analysis suggested the predominance of both disordered and β-strands regions in the secondary structure. CaTI1 presented striking physico-chemical stability. In an attempt to get the entire sequence of CaTI1 we found another PI called CaTI2. The discussion of this finding is in the main text. A degenerate primer was designed based on the sequence of trypsin inhibitor CaTI1 in an attempt to achieve the cloning of this PI. Surprisingly, the alignment of the predicted peptide derived from the cDNA with the protein database showed similarity with other C. annuun PIs, and thus it was called CaTI3.

Diacylglycerol acyltransferase 1 (DGAT1) is a microsomal membrane enzyme responsible for the final step in the synthesis of triacylglycerides. Although DGATs from a wide range of organisms have nearly identical sequences, there is little structural information available for these enzymes. The substrate binding sites of DGAT1 are predicted to be in its large luminal extramembranous loop and to include common motifs with acyl-CoA cholesterol acyltransferase enzymes and the diacylglycerol binding domain found in protein kinases.In this study, synthetic peptides corresponding to the predicted binding sites of DGAT1 enzyme were examined using synchrotron radiation circular dichroism spectroscopy, fluorescence emission and adsorption onto lipid monolayers to determine their interactions with substrates associated with triacylglyceride synthesis (oleoyl-CoA and dioleoylglycerol). One of the peptides, Sit1, which includes the FYxDWWN motif common to both DGAT1 and acyl-CoA cholesterol acyltransferase, changes its conformation in the presence of both substrates, suggesting its capability to bind their acyl chains. The other peptide (Sit2), which includes the putative diacylglycerol binding domain HKWCIRHFYKP found in protein kinase C and diacylglycerol kinases, appears to interact with the charged headgroup region of the substrates. Moreover, in an extended-peptide which contains Sit1 and Sit2 sequences separated by a flexible linker, larger conformational changes were induced by both substrates, suggesting that the two binding sites may bring the substrates into close proximity within the membrane, thus catalyzing the formation of the triacylglyceride product.

Plantaricin149a (Pln149a) is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD) of the antimicrobial peptide from the liposomes (approximately three-fold higher), and decreased the enthalpy of binding to anionic vesicles from −17.2 kcal/mol to −10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface., The authors thank Heloisa S.S Araújo (UFSCar, Brazil) for peptide sequencing. This work was supported by grants from FAPESP and CNPq (Brazilian agencies) and grant from PRPG-USP/Santander (J.L.S.L.).

Store-operated calcium entry (SOCE) is a conserved calcium pathway in eukaryotes, although not yet studied in Schistosoma mansoni, the main causative agent of schistosomiasis in America. The proteins STIM1 (calcium sensor) and Orai1 (calcium channel) are the central components of SOCE and the recently published genome of this parasite encodes orthologs of these two proteins, indicating that this Ca2+ pathway is present in S. mansoni.STIM comprises several domains, including three cytosolic coiled coil domains (CC). The first CC (STIM_1CC) is predicted to interact with the other two CC domains in the resting state, and upon activation by calcium depletion, the channel activating domain (CAD), comprised by the second and third CC, is exposed to interact with the C-terminus of Orai, opening the channel.The aim of the present work is to characterize the SOCE pathway in S. mansoni, through the expression of the recombinant proteins from this pathway and interaction assays among STIM and Orai domains, in order to get insights into the mechanism of action of this calcium channel in platyhelminths.S. mansoni STIM_1CC (SmSTIM_1CC), SmSTIM_CAD and SmOrai-C were obtained by recombinant expression in E. coli. Pull down and Isotermic Titration calorimetry experiments showed that SmOrai-C interacts with SmSTIM_CAD in vitro, indicating that the SOCE pathway might be functional in Schistosomes. Additionally, pull down and surface plasmon resonance experiments indicated that SmSTIM_1CC and SmSTIM_CAD also interact with each other, which may consist in a mechanism of autoinhibition of the channel in the resting state, as reported for other organisms. Therefore, the mechanisms by which SmStim activates SmOrai appear highly conserved and the characterization of a new calcium channel for this important human parasite sheds light into possible new targets against schistosomiasis.

The molecular mechanisms responsible for protein structural changes in the central nervous system leading to Alzheimer's disease are unknown, but there is evidence that a family of proteins known as septins may be involved. Septins are a conserved group of GTP-binding proteins which participate in various cellular processes, including polarity determination and membrane dynamics. SEPT1, SEPT4, and SEPT2 have been found in deposits known as neurofibrillary tangles and glial fibrils in Alzheimer's disease. In this study, we provide molecular-level information for the interaction of SEPT2 with Langmuir monolayers at the air/water interface, which are used as simplified membrane models. The high surface activity of SEPT2 causes it to adsorb onto distinct types of lipid Langmuir monolayers, namely dipalmitoylphosphatidylcholine and PtdIns(4,5)P2. However, the interaction with PtdIns(4,5)P2 is much stronger, not only leading to a higher adsorption, but also to SEPT2 remaining inserted within the membrane at high surface pressures. Most importantly, in situ polarization-modulated infrared reflection absorption spectroscopy results indicated that the native secondary structure of SEPT2 is preserved upon interacting with PtdIns(4,5)P2, but not when dipalmitoylphosphatidylcholine is at the air/water interface. Taken together, the results presented here suggest that the interaction between SEPT2 and the cell membrane may play an important role in the assembly of SEPT2 into amyloid-like fibers.

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).

The micro-exon genes (MEG) identified in the genome of Schistossoma mansoni, a flatworm parasite responsible for the second most widespread tropical disease, code for a class of small secreted proteins that are highly expressed in the human host stage. Due to the high variability in primary structure of the MEG proteins, the parasite is thought to recruit them to trick the host immune system; however, their specific function is still not understood. We have examined the secondary structure of MEG14, a member of this family, as well as the effects of a range of physical-chemical factors on its conformation, using synchrotron radiation circular dichroism (SRCD) spectroscopy and Oriented-SRCD. The SRCD spectrum of MEG14 in aqueous solution exhibits a strong minimum at 198 nm and a small positive band at 182 nm, corresponding a high content of disordered structure (>80%). However, disorder-to-order transitions that lead to the induction of an alpha-helix structure in MEG14 were noted by the addition of trifluoroethanol (≥ 50%) with negative peak at 208 and 222 nm and a stronger positive peak at 192 with a shoulder at 175 nm. This ordering effect was also produced by increasing temperature (in a reversible process), the presence of negatively charged lipids (like POPG and SDS), and absorption on a silica surface, followed by dehydration. Taken together with consideration of its primary structure (high content of charged and proline residues, low content of aromatic and/or hydrophobic residues), these results support the classification of MEG14 as good model of an intrinsically disordered protein, and suggest the possibility of its interaction with different partners in the host to produce multifunctional roles.(Support by grants from FAPESP, CNPq, MCTI, and the U.K. BBRSC, and beamtime from the ANKA, ISA and Soleil Synchrotrons)

The action of a synthetic antimicrobial peptide analog of Plantaricin 149 (Pln149a) against Saccharomyces 24 ^ cerevisiae and its interaction with biomembrane model systems were investigated. Pln149a was shown to 25 inhibit S. cerevisiae growth by more than 80% in YPD medium, causing morphological changes in the yeast 26 wall and remaining active and resistant to the yeast proteases even after 24 h of incubation. Different 27 membrane model systems and carbohydrates were employed to better describe the Pln149a interaction with 28 cellular components using circular dichroism and fluorescence spectroscopies, adsorption kinetics and 29 surface elasticity in Langmuir monolayers. These assays showed that Pln149a does not interact with either 30 mono/polysaccharides or zwitterionic LUVs, but is strongly adsorbed to and incorporated into negatively 31 charged surfaces, causing a conformational change in its secondary structure from random-coil to helix upon 32 adsorption. From the concurrent analysis of Pln149a adsorption kinetics and dilatational surface elasticity 33 data, we determined that 2.5 μM is the critical concentration at which Pln149a will disrupt a negative DPPG 34 monolayer. Furthermore, Pln149a exhibited a carpet-like mechanism of action, in which the peptide initially 35 binds to the membrane, covering its surface and acquiring a helical structure that remains associated to the 36 negatively charged phospholipids. After this electrostatic interaction, another peptide region causes a strain 37 in the membrane, promoting its disruption. Fil: Lopes, José Luiz S.. Universidade de Sao Paulo; Brasil Fil: Nobre, Thatyane M.. Universidade de Sao Paulo; Brasil Fil: Siano, Alvaro Sebastían. Universidad Nacional del Litoral; Argentina Fil: Humpola, Verónica. Universidad Nacional del Litoral; Argentina Fil: Bossolan, Nelma R.S.. Universidade de Sao Paulo; Brasil Fil: Zaniquelli, Maria. E.D.. Universidade de Sao Paulo; Brasil Fil: Tonarelli, Georgina Guadalupe. Universidad Nacional del Litoral; Argentina Fil: Beltramini, Leila M.. Universidade de Sao Paulo; Brasil

This study aimed at investigating the structural properties and mechanisms of the antifungal action of CpOsm, a purified osmotin from Calotropis procera latex. Fluorescence and CD assays revealed that the CpOsm structure is highly stable, regardless of pH levels. Accordingly, CpOsm inhibited the spore germination of Fusarium solani in all pH ranges tested. The content of the secondary structure of CpOsm was estimated as follows: α-helix (20%), β-sheet (33%), turned (19%) and unordered (28%), RMSD 1%. CpOsm was stable at up to 75 °C, and thermal denaturation (T m ) was calculated to be 77.8 °C. This osmotin interacted with the negatively charged large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-1-glycerol (POPG), inducing vesicle permeabilization by the leakage of calcein. CpOsm induced the membrane permeabilization of spores and hyphae from Fusarium solani , allowing for propidium iodide uptake. These results show that CpOsm is a stable protein, and its antifungal activity involves membrane permeabilization, as property reported earlier for other osmotins and thaumatin-like proteins.

Circular dichroism spectroscopy is a well-used method for studying the secondary structure of proteins in solution. Synchrotron radiation circular dichroism (SRCD) offers the advantage of improving accuracy, due to the additional low wavelength data that can be measured.Intrinsically disordered proteins (IDPs), which lack significant amounts of their structure in canonical secondary structures such as helixes, sheets, or turns, are an important class of protein that have not, to date, been accurately analysed by circular dichroism spectroscopy because of the lack of an appropriate reference data base of proteins available for empirical analyses. Furthermore, the “other” category, often considered to represent disordered structures generally also includes polyproline-II (PPII) structures, because until now their spectral properties have not been well-distinguished from unordered structures.In this study, SRCD spectroscopy was used to characterize the spectral properties of PPII structures, using collagen and polyproline as representatives of this type of structure. The spectra of the polypeptides were obtained under “native” and a variety of denaturing conditions, such as thermal unfolding and chemical unfolding (by the presence of chaotropic or reducing agents, pH or detergents, and dehydration), with the aim of defining the differences between polyproline and disordered structures.The single most characteristic spectroscopic feature distinguishing the spectra of polyproline and unordered structures was found to be a positive peak around 220 nm in the former but not in the latter. As a consequence this data is being used, in addition to new spectra on IDPs to create a improved reference data set for the empirical analyses of IDPs, and PPII structures.Supported by: BBSRC, and CNPq grants and beamtime grants from ANKA, ISA, BSRF, and Soleil synchrotrons (to BAW and/or JLSL).

Proteins belonging to the S100 family regulate many cellular functions, including cell growth, differentiation, motility, contraction and intracellular signal transduction. S100A12 is a small calcium-binding protein highly abundant in the cytosol of granulocytes, where it has a possible role in signal modulation of inflammatory processes.In this study, the binding of porcine S100A12 (Calgranulin C) to lipid bilayers and the effect of ions (calcium and zinc) in modulating its interaction with liposomes was investigated using synchrotron radiation circular dichroism (SRCD), fluorescence emission and surface plasmon resonance spectroscopies.The binding of S100A12 to phospholipid vesicles occurred both in its apo- and holo- forms, however the protein was bound more tightly to negatively-charged liposomes. Moreover, the presence of the ions facilitated its interaction with liposomes, producing distinct conformational changes and severely reducing its thermal stability.These S100A12-lipid interactions may be a way of translating physiological changes in calcium/zinc levels into specific cellular responses. In addition S100A12 may exist in a dynamic exchange between cytosolic and membrane-associated states, regulated by specific cellular signals.(Support by grants from FAPESP, CNPq and the BBRSC, and beamtime from the ISA Synchrotron facility, Denmark).