A Fourier transform infrared spectroscopy study of cell membrane domain modifications induced by docosahexaenoic acid.

BACKGROUND: Detergent resistant membranes (DRMs) are a useful model system for the in vitro characterization of cell membrane domains. Indeed, DRMs provide a simple model to study the mechanisms underlying several key cell processes based on the interplay between specific cell membrane domains on one hand, and specific proteins and/or lipids on the other. Considering therefore their biological relevance, the development of methods capable to provide information on the composition and structure of membrane domains and to detect their modifications is highly desirable. In particular, Fourier transform infrared (FTIR) spectroscopy is a vibrational tool widely used for the study not only of isolated and purified biomolecules but also of complex biological systems, including intact cells and tissues. One of the main advantages of this non-invasive approach is that it allows obtaining a molecular fingerprint of the sample under investigation in a rapid and label-free way. METHODS: Here we present an FTIR characterization of DRM fractions purified from the human breast cancer cells MCF-7, before and after treatment with the omega 3 fatty acid docosahexaenoic acid (DHA), which was found to promote membrane microdomain reorganization. RESULTS AND CONCLUSIONS: We will show that FTIR spectroscopy coupled with multivariate analysis enables to monitor changes in the composition of DRMs, induced in particular by the incorporation of DHA in cell membrane phospholipids. GENERAL SIGNIFICANCE: This study paves the way for a new label-free characterization of specific membrane domains within intact cells, which could provide complementary information to the fluorescence approaches presently used.

Why and how protein aggregation has to be studied in vivo.

The understanding of protein aggregation is a central issue in different fields of protein science, from the heterologous protein production in biotechnology to amyloid aggregation in several neurodegenerative and systemic diseases. To this goal, it became more and more evident the crucial relevance of studying protein aggregation in the complex cellular environment, since it allows to take into account the cellular components affecting protein aggregation, such as chaperones, proteases, and molecular crowding. Here, we discuss the use of several biochemical and biophysical approaches that can be employed to monitor protein aggregation within intact cells, focusing in particular on bacteria that are widely employed as microbial cell factories.

Effects of methanol on a methanol-tolerant bacterial lipase.

Methanol is often employed in biocatalysis with the purpose of increasing substrates solubility or as the acyl acceptor in transesterification reactions, but inhibitory effects are observed in several cases. We have studied the influence of methanol on the catalytic activity and on the conformation of the lipase from Burkholderia glumae, which is reported to be highly methanol tolerant if compared with other lipases. We detected highest activity in the presence of 50-70 % methanol. Under these conditions, however, the enzyme stability is perturbed, leading to gradual protein unfolding and finally to aggregation. These results surmise that, for this lipase, methanol-induced deactivation does not depend on inhibition of catalytic activity but rather on negative effects on the conformational stability of the catalyst.

Structure, stability, and aggregation of ß-2 microglobulin mutants: insights from a Fourier transform infrared study in solution and in the crystalline state.

ß-2 microglobulin (ß2m) is an amyloidogenic protein involved in dialysis-related amyloidosis. We report here the study of the structural properties of the protein in solution and in the form of single crystals by Fourier transform infrared (FTIR) spectroscopy and microspectroscopy. The investigation has been extended to four ß2m mutants previously characterized by x-ray crystallography: Asp(53)Pro, Asp(59)Pro, Trp(60)Gly, and Trp(60)Val. These variants displayed very similar three-dimensional structures but different thermal stability and aggregation propensity, investigated here by FTIR spectroscopy. For each variant, appreciable spectral differences were found between the protein in solution and in single crystals, consisting in a downshift of the main ß-sheet band and in better resolved turn and loop bands, indicative of reduced protein secondary structure dynamics in the crystalline state. Notably, the well-resolved spectra of the ß2m crystalline variants enabled us to identify structural differences induced by the single amino acid mutations. Such differences encompass turn and loop structures that might affect the stability and aggregation propensity of the investigated ß2m variants. This study highlights the potential of FTIR microspectroscopy to acquire useful structural information on protein crystals, complementary to the crystallographic analyses.

FTIR spectral signatures of mouse antral oocytes: molecular markers of oocyte maturation and developmental competence.

Mammalian antral oocytes with a Hoescht-positive DNA ring around the nucleolus (SN) are able to resume meiosis and to fully support the embryonic development, while oocytes with a non-surrounded nucleolus (NSN) cannot. Here, we applied FTIR microspectroscopy to characterize single SN and NSN mouse oocytes in order to try to elucidate some aspects of the mechanisms behind the different chromatin organization that impairs the full development of NSN oocyte-derived embryos. To this aim, oocytes were measured at three different stages of their maturation: just after isolation and classification as SN and NSN oocytes (time 0); after 10h of in vitro maturation, i.e. at the completion of the metaphase I (time 1); and after 20h of in vitro maturation, i.e. at the completion of the metaphase II (time 2). Significant spectral differences in the lipid (3050-2800cm(-1)) and protein (1700-1600cm(-1)) absorption regions were found between the two types of oocytes and among the different stages of maturation within the same oocyte type. Moreover, dramatic changes in nucleic acid content, concerning mainly the extent of transcription and polyadenylation, were detected in particular between 1000 and 800cm(-1). The use of the multivariate principal component-linear discriminant analysis (PCA-LDA) enabled us to identify the maturation stage in which the separation between the two types of oocytes took place, finding as the most discriminating wavenumbers those associated to transcriptional activity and polyadenylation, in agreement with the visual analysis of the spectral data.

Fluorescence of KCl Aqueous Solution: A Possible Spectroscopic Signature of Nucleation.

Ion pairing in water solutions alters both the water hydrogen-bond network and ion solvation, modifying the dynamics and properties of electrolyte water solutions. Here, we report an anomalous intrinsic fluorescence of KCl aqueous solution at room temperature and show that its intensity increases with the salt concentration. From the ab initio density functional theory (DFT) and time-dependent DFT modeling, we propose that the fluorescence emission could originate from the stiffening of the hydrogen bond network in the hydration shell of solvated ion-pairs that suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway. Because computations suggest that the fluorophores are the local ion-water structures present in the prenucleation phase, this band could be the signature of the incoming salt precipitation.

Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain.

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.

Compact conformations of α-synuclein induced by alcohols and copper.

The intrinsically disordered protein α-synuclein aggregates into amyloid fibrils, a process known to be implicated in several neurodegenerative states. Partially folded forms of the protein are thought to trigger the aggregation process. Here, α-synuclein conformers are characterized by analysis of the charge-state distributions observed in electrospray-ionization mass spectrometry under negative-ion mode. It is found that, even at neutral pH, a small fraction of the molecular population is in a compact conformation. Several distinct partially folded forms are then identified under conditions that promote α-synuclein aggregation, such as solutions of simple and fluorinated alcohols. Specific intermediates accumulate at increasing concentrations of ethanol, hexafluoro-2-propanol, and trifluoroethanol. Finally, extensive folding induced by Cu(2+) binding is revealed by titrations in the presence of Cu(2+)-glycine. The data confirm the existence of a single, high-affinity binding site for Cu(2+). Because accumulation of this partially folded form correlates with enhancement of fibrillation kinetics, it is likely to represent an amyloidogenic intermediate in α-synuclein conformational transitions.

Tetracycline prevents Aß oligomer toxicity through an atypical supramolecular interaction.

The antibiotic tetracycline was reported to possess an anti-amyloidogenic activity on a variety of amyloidogenic proteins both in in vitro and in vivo models. To unveil the mechanism of action of tetracycline on Aß1-40 and Aß1-42 at both molecular and supramolecular levels, we carried out a series of experiments using NMR spectroscopy, FTIR spectroscopy, dynamic laser light-scattering (DLS) and atomic force microscopy (AFM). Firstly we showed that the co-incubation of Aß1-42 oligomers with tetracycline hinders the toxicity towards N2a cell lines in a dose-dependent manner. Therefore, the nature of the interaction between the drug and Aß oligomers was investigated. To carry out NMR and FTIR studies we have prepared Aß peptide solutions containing assemblies ranging from monomers to large oligomers. Saturation transfer difference (STD) NMR experiments have shown that tetracycline did not interact with monomers at variance with oligomers. Noteworthy, in this latter case we observed that this interaction was very peculiar since the transfer of magnetization from Aß oligomers to tetracycline involved all drug protons. In addition, intermolecular cross-peaks between tetracycline and Aß were not observed in NOESY spectra, indicating the absence of a specific binding site and suggesting the occurrence of a supramolecular interaction. DLS and AFM studies supported this hypothesis since the co-dissolution of Aß peptides and tetracycline triggered the immediate formation of new aggregates that improved the solubility of Aß peptides, preventing in this way the progression of the amyloid cascade. Moreover, competitive NMR binding experiments showed for the first time that tetracycline competes with thioflavin T (ThT) in the binding to Aß peptides. Our data shed light on a novel mechanism of anti-amyloidogenic activity displayed by tetracycline, governed by hydrophobic and charge multiparticle interactions.

Tetracycline and its analogues protect Caenorhabditis elegans from ß amyloid-induced toxicity by targeting oligomers.

The accumulation and deposition of amyloid beta (Aß) peptide in extracellular dense plaques in the brain is a key phase in Alzheimer's disease (AD). Small oligomeric forms of Aß are responsible for the toxicity and the early cognitive impairment observed in patients before the amyloid plaque deposits appear. It is essential for the development of an efficient cure for AD to identify compounds that interfere with Aß aggregation, counteracting the molecular mechanisms involved in conversion of the monomeric amyloid protein into oligomeric and fibrillar forms. Tetracyclines have been proposed for AD therapy, although their effects on the aggregation of Aß protein, particularly their ability to interact in vivo with the Aß oligomers and/or aggregates, remain to be understood. Using transgenic Caenorhabditis elegans as a simplified invertebrate model of AD, we evaluated the ability of tetracyclines to interfere with the sequence of events leading to Aß proteotoxicity. The drugs directly interact with the Aß assemblies in vivo and reduce Aß oligomer deposition, protecting C. elegans from oxidative stress and the onset of the paralysis phenotype. These effects were specific, dose-related and not linked to any antibiotic activity, suggesting that the drugs might offer an effective therapeutic strategy to target soluble Aß aggregates.

Embryonic stem cell differentiation studied by FT-IR spectroscopy.

We propose, here, an FT-IR method to monitor the spontaneous differentiation of murine embryonic stem (ES) cells in their early development. Principal component analysis and subsequent linear discriminant analysis enabled us to segregate stem cell spectra into separate clusters - corresponding to different differentiation times - and to identify the most significant spectral changes during differentiation. Between days 4 to 7 of differentiation, these spectral changes in the protein amide I band (1700-1600 cm(-1)) and in the nucleic acid absorption region (1050-850 cm(-1)) indicated that mRNA translation was taking place and that specific proteins were produced, reflecting the appearance of a new phenotype. The DNA/RNA hybrid bands (954 cm(-1) and 899 cm(-1)) were also observed, suggesting that the transcriptional switch of the genome started at this stage of differentiation. As confirmed by cytochemical assays, the FT-IR approach presented here allows to detect at molecular level the biological events of ES cell differentiation as they take place and to monitor in a rapid way the temporal evolution of the ES cell culture.

The osmolyte betaine promotes protein misfolding and disruption of protein aggregates.

In this work the effect of betaine on the structure and aggregation of the GST-GFP fluorescent fusion protein was studied by different complementary techniques, including electron microscopy, dynamic light scattering, circular dichroism, and FTIR spectroscopy. Although osmolytes are known to be protein stabilizers in vivo, the effect of betaine on the structure and aggregation of our model protein was found to be strictly concentration dependent. We demonstrated that, by changing betaine concentration, it was possible to tune the formation of protein soluble assemblies and insoluble aggregates, as well as to disaggregate preformed aggregates. In particular, at a critical concentration of betaine between 5 and 7.5 mM, the protein precipitated into macroscopic prefibrillar structures, rich in intermolecular beta-sheets, which were found to bind thioflavine T and to be inaccessible to protease. Instead, at higher betaine concentration (10-20 mM) the misfolded protein lost its fluorescence, but formed soluble assemblies with hydrodynamic radius of about 16 nm. These structures displayed a reduced propensity to further aggregate under thermal treatment. In addition, betaine at this high concentration was also found to disrupt large preformed aggregates, obtained under different conditions, into protein soluble assemblies. It is the first time that a disaggregation process has been described for a chemical chaperone. A mechanism for the betaine concentration-dependent effect on protein misfolding, aggregation, and disaggregation is proposed and its possible physiological implications are discussed.

Anomalous Intrinsic Fluorescence of HCl and NaOH Aqueous Solutions.

The unique properties of liquid water mainly arise from its hydrogen bond network. The geometry and dynamics of this network play a key role in shaping the characteristics of soft matter, from simple solutions to biosystems. Here we report an anomalous intrinsic fluorescence of HCl and NaOH aqueous solutions at room temperature that shows important differences in the excitation and emission bands between the two solutes. From ab initio time-dependent density functional theory modeling we propose that fluorescence emission could originate from hydrated ion species contained in transient cavities of the bulk solvent. These cavities, which are characterized by a stiff surface, could provide an environment that, upon trapping the excited state, suppresses the fast nonradiative decay and allows the slower radiative channel to become a possible decay pathway.

Alpha-helix stabilization within a peptide dendrimer.

The alpha-helical second generation peptide dendrimer of sequence (AcAMEA)(4)(KKLME)(2)KMKLA is more stable than the corresponding linear peptide AcAMEAAKLMEAMKLA toward pH-induced unfolding and temperature-induced intermolecular aggregation. The effect is interpreted in terms of an alpha-helix spanning across two successive branching points of the dendrimer. This stabilization effect is unprecedented and opens the way to folded dendritic analogues of proteins using natural amino acids only.

In situ protein folding and activation in bacterial inclusion bodies.

Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.

Physical and chemical perturbations induce the formation of protein aggregates with different structural features.

The in vitro aggregation of the model GST-GFP fusion protein was induced by several effectors, including those mimicking variations occurring under cell stress conditions. In particular, we examined the effects of thermal treatments, redox state and pH variations, salt addition, and freezing and thawing cycles. The resulting aggregates displayed different morphologies as seen by electron microscopy, and different secondary and tertiary structures, as indicated by Fourier transform infrared spectroscopy and fluorescence. Therefore, proteins can be forced to undergo multiple aggregation pathways that lead to assemblies with different molecular structures and, possibly, specific physiological and pathological roles. In conclusion, great caution should be taken in inferring conclusions on protein aggregation and disaggregation in vivo from results obtained using aggregates produced under non-physiological perturbations.

In vivo aggregation of bovine beta-lactoglobulin is affected by Cys at position 121.

Bovine beta-lactoglobulin (BLG) has been widely used as a model system to study protein folding and aggregation and for biotechnology applications. Native BLG contains two disulfide bonds and one free cysteine at position 121. This free thiol group has been shown to be responsible for the irreversibility of BLG denaturation in vitro, but nothing is known about its relevance during protein folding inside the cell. Here, we report the expression of soluble wild type recombinant BGL in Escherichia coli cells at about 109 mg rBLG/g wet weight cells and a comparison between the aggregation of wt BLG and its variant C121S upon intracellular expression. We show that in E. coli C121SBLG is more prone to aggregation than the wild type protein and that their different behavior depends on the oxidation of disulfide bonds. Our results underline the key contribution of the unpaired cysteine residue during the oxidative folding pathway and indicate BLG as a useful tool for the study of protein aggregation in vivo.

A FTIR microspectroscopy study of the structural and biochemical perturbations induced by natively folded and aggregated transthyretin in HL-1 cardiomyocytes.

Protein misfolding and aggregation are associated with a number of human degenerative diseases. In spite of the enormous research efforts to develop effective strategies aimed at interfering with the pathogenic cascades induced by misfolded/aggregated peptides/proteins, the necessary detailed understanding of the molecular bases of amyloid formation and toxicity is still lacking. To this aim, approaches able to provide a global insight in amyloid-mediated physiological alterations are of importance. In this study, we exploited Fourier transform infrared microspectroscopy, supported by multivariate analysis, to investigate in situ the spectral changes occurring in cultured intact HL-1 cardiomyocytes exposed to wild type (WT) or mutant (L55P) transthyretin (TTR) in native, or amyloid conformation. The presence of extracellular deposits of amyloid aggregates of WT or L55P TTR, respectively, is a key hallmark of two pathological conditions, known as senile systemic amyloidosis and familial amyloid polyneuropathy. We found that the major effects, associated with modifications in lipid properties and in the cell metabolic/phosphorylation status, were observed when natively folded WT or L55P TTR was administered to the cells. The effects induced by aggregates of TTR were milder and in some cases displayed a different timing compared to those elicited by the natively folded protein.

Two-dimensional versus three-dimensional morphometry of monogenoidean sclerites.

A new method of three-dimensional (3-D) analysis of sclerotised structures of monogenoids was performed by processing z-series images using 3D-Doctor. Z-series were obtained from Gomori's trichrome-stained specimens of marine and freshwater monogenoids under laser scanning confocal fluorescence microscopy. Measurements obtained from 3-D images were then compared with those from 2-D images taken from both flattened and unflattened specimens. Data comparison demonstrated that 3-D morphometry allowed avoidance of over-estimation due to deformation and the reduction of errors associated with different spatial orientations. Moreover, study of 3-D images permitted observation of morphological details that are not detectable in 2-D representations.

Chemical space guided discovery of antimicrobial bridged bicyclic peptides against Pseudomonas aeruginosa and its biofilms.

Herein we report the discovery of antimicrobial bridged bicyclic peptides (AMBPs) active against Pseudomonas aeruginosa, a highly problematic Gram negative bacterium in the hospital environment. Two of these AMBPs show strong biofilm inhibition and dispersal activity and enhance the activity of polymyxin, currently a last resort antibiotic against which resistance is emerging. To discover our AMBPs we used the concept of chemical space, which is well known in the area of small molecule drug discovery, to define a small number of test compounds for synthesis and experimental evaluation. Our chemical space was calculated using 2DP, a new topological shape and pharmacophore fingerprint for peptides. This method provides a general strategy to search for bioactive peptides with unusual topologies and expand the structural diversity of peptide-based drugs.