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We consider an unsupervised bilevel optimization strategy for learning regularization parameters in the context of imaging inverse problems in the presence of additive white Gaussian noise. Compared to supervised and semi-supervised metrics relying either on the prior knowledge of reference data and/or on some (partial) knowledge on the noise statistics, the proposed approach optimizes the whiteness of the residual between the observed data and the observation model with no need of ground-truth data.We validate the approach on standard Total Variation-regularized image deconvolution problems which show that the proposed quality metric provides estimates close to the mean-square error oracle and to discrepancy-based principles.

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.

The tear film is a complex matrix composed of several molecular classes, from small metal ions to macromolecules. Contact lens (CL) wear can affect the protein homeostasis of the tear film, by accumulating deposits on the CL surface and/or altering their structural and functional properties. This work investigates the effect of CL wear on lactoferrin (Lf), one of the most abundant tear proteins, known as an unspecific biomarker of inflammation. Tears from eight volunteers were collected and analyzed after alternated periods of CL wear and without CL. The experimental approach is to probe Lf into unprocessed human tears by the peculiar fluorescence emission originating from complex formation of Lf with terbium (Tb3+) at the iron-binding sites. The experimental data indicate that CL wear does not significantly affect the total amount of Lf. On the other hand, Lf affinity for Tb3+ is reduced upon CL wear, suggesting relevant changes in Lf structure and possible alterations of protein functionality. Future studies based on this approach will help define CL features (material, lens-care solution, wearing time, etc.) with minimal effects on tear protein activity, in order to obtain more biocompatible and comfortable devices.

The lacrimal film has attracted increasing interest in the last decades as a potential source of biomarkers of physiopathological states, due to its accessibility, moderate complexity, and responsiveness to ocular and systemic diseases. High-performance liquid chromatography-mass spectrometry (LC-MS) has led to effective approaches to tear proteomics, despite the intrinsic limitations in sample amounts. This review focuses on the recent progress in strategy and technology, with an emphasis on the potential for personalized medicine. After an introduction on lacrimal-film composition, examples of applications to biomarker discovery are discussed, comparing approaches based on pooled-sample and single-tear analysis. Then, the most critical steps of the experimental pipeline, that is, tear collection, sample fractionation, and LC-MS implementation, are discussed with reference to proteome-coverage optimization. Advantages and challenges of the alternative procedures are highlighted. Despite the still limited number of studies, tear quantitative proteomics, including single-tear investigation, could offer unique contributions to the identification of low-invasiveness, sustained-accessibility biomarkers, and to the development of personalized approaches to therapy and diagnosis.

Intrinsically disordered proteins (IDPs) are ensembles of interconverting conformers whose conformational properties are governed by several physico-chemical factors, including their amino acid composition and the arrangement of oppositely charged residues within the primary structure. In this work, we investigate the effects of charge patterning on the average compactness and shape of three model IDPs with different proline content. We model IDP ensemble conformations as ellipsoids, whose size and shape are calculated by combining data from size-exclusion chromatography and native mass spectrometry. For each model IDP, we analyzed the wild-type protein and two synthetic variants with permuted positions of charged residues, where positive and negative amino acids are either evenly distributed or segregated. We found that charge clustering induces remodeling of the conformational ensemble, promoting compaction and/or increasing spherical shape. Our data illustrate that the average shape and volume of the ensembles depend on the charge distribution. The potential effect of other factors, such as chain length, number of proline residues, and secondary structure content, is also discussed. This methodological approach is a straightforward way to model IDP average conformation and decipher the salient sequence attributes influencing IDP structural properties.

The lacrimal film has attracted increasing interest in the last decades as a potential source of biomarkers of physiopathological states, due to its accessibility, moderate complexity, and responsiveness to ocular and systemic diseases. High-performance liquid chromatography-mass spectrometry (LC-MS) has led to effective approaches to tear proteomics, despite the intrinsic limitations in sample amounts. This review focuses on the recent progress in strategy and technology, with an emphasis on the potential for personalized medicine. After an introduction on lacrimal-film composition, examples of applications to biomarker discovery are discussed, comparing approaches based on pooled-sample and single-tear analysis. Then, the most critical steps of the experimental pipeline, that is, tear collection, sample fractionation, and LC-MS implementation, are discussed with reference to proteome-coverage optimization. Advantages and challenges of the alternative procedures are highlighted. Despite the still limited number of studies, tear quantitative proteomics, including single-tear investigation, could offer unique contributions to the identification of low-invasiveness, sustained-accessibility biomarkers, and to the development of personalized approaches to therapy and diagnosis.

Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB., QC 20200622

Intrinsically disordered proteins (IDPs) are systematically under-represented in structural proteomics studies. Their structural characterization implies description of the dynamic conformational ensembles populated by these polymers in solution, posing major challenges to biophysical methods. “Native” MS (native-MS) has emerged as a central tool in this field, conjugating the unique MS analytical power with structurally meaningful descriptors, like solvent-accessible surface area (SASA) and collisional cross section (CCS). This review summarizes recently published papers comparing native-MS and solution methods, with a focus on charge-state-distribution (CSD) analysis for IDP conformational analysis. The results point to substantial agreement, supporting structural interpretation of native-MS spectra of IDPs. The discussion is integrated with data from our group on “synthetic” IDPs, obtained by reduction and alkylation of natively folded proteins, whose fold is stabilized by disulfide bridges. Finally, an MS-based compaction index (CI) is introduced, evaluating SASA with reference to globular and fully disorder proteins. Such a parameter can be calculated for single conformers or the whole conformational ensemble, offering a continuous index for IDP comparison and classification.

Metal nanoclusters featuring tunable luminescence and high biocompatibility are receiving attention as fluorescent markers for cellular imaging. The recently discovered ability of gold clusters to scavenge cytotoxic reactive oxygen species (ROS) from the intracellular environment extends their applicability to biomedical theranostics and provides a novel platform for realizing multifunctional luminescent probes with engineered anti-cytotoxic activity for applications in bio-diagnostics and conceivably cellular therapy. This goal could be achieved by using clusters of strongly reactive metals such as silver, provided that strategies are found to enhance their luminescence while simultaneously enabling direct interaction between the metal atoms and the chemical surroundings. In this work, we demonstrate a synergic approach for realizing multifunctional metal clusters combining enhanced luminescence with strong and lasting ROS scavenging activity, based on the fabrication and in situ protection of Ag nanoclusters with a supramolecular mantle of thiolated-Au atoms (Ag/Au-t). Confocal imaging and viability measurements highlight the biocompatibility of Ag/Au-t and their suitability as fluorescent bio-markers. ROS concentration tests reveal the remarkable scavenging activity of Ag-based clusters. Proliferation tests of cells in artificially stressed culture conditions point out their prolonged anti-cytotoxic effect with respect to gold systems, ensuring positive cell proliferation rates even for long incubation time.

Metal nanoclusters featuring tunable luminescence and high biocompatibility are receiving attention as fluorescent markers for cellular imaging. The recently discovered ability of gold clusters to scavenge cytotoxic reactive oxygen species (ROS) from the intracellular environment extends their applicability to biomedical theranostics and provides a novel platform for realizing multifunctional luminescent probes with engineered anti-cytotoxic activity for applications in bio-diagnostics and conceivably cellular therapy. This goal could be achieved by using clusters of strongly reactive metals such as silver, provided that strategies are found to enhance their luminescence while simultaneously enabling direct interaction between the metal atoms and the chemical surroundings. In this work, we demonstrate a synergic approach for realizing multifunctional metal clusters combining enhanced luminescence with strong and lasting ROS scavenging activity, based on the fabrication and in situ protection of Ag nanoclusters with a supramolecular mantle of thiolated-Au atoms (Ag/Au-t). Confocal imaging and viability measurements highlight the biocompatibility of Ag/Au-t and their suitability as fluorescent bio-markers. ROS concentration tests reveal the remarkable scavenging activity of Ag-based clusters. Proliferation tests of cells in artificially stressed culture conditions point out their prolonged anti-cytotoxic effect with respect to gold systems, ensuring positive cell proliferation rates even for long incubation time.

The synthesis of novel hyperbranched monodisperse linear dendrimers, based on 2,2-bis-(hydroxymethyl)-propionic acid (bis-MPA), has been achieved by convergent metathesis-mediated coupling between the alkene-terminated focal point of bis-MPA dendrons. On their surface, dendrimers present 4, 8 and 16 functional groups. Glycodendrimers exposing multiple saccharide moieties have also been obtained. To the best of our knowledge, this is the first example of the use of metathesis for focal point coupling.

Native mass spectrometry (MS) has become a central tool of structural proteomics, but its applicability to the peculiar class of intrinsically disordered proteins (IDPs) is still object of debate. IDPs lack an ordered tridimensional structure and are characterized by high conformational plasticity. Since they represent valuable targets for cancer and neurodegeneration research, there is an urgent need of methodological advances for description of the conformational ensembles populated by these proteins in solution. However, structural rearrangements during electrospray-ionization (ESI) or after the transfer to the gas phase could affect data obtained by native ESI-MS. In particular, charge-state distributions (CSDs) are affected by protein conformation inside ESI droplets, while ion mobility (IM) reflects protein conformation in the gas phase. This review focuses on the available evidence relating IDP solution ensembles with CSDs, trying to summarize cases of apparent consistency or discrepancy. The protein-specificity of ionization patterns and their responses to ligands and buffer conditions suggests that CSDs are imprinted to protein structural features also in the case of IDPs. Nevertheless, it seems that these proteins are more easily affected by electrospray conditions, leading in some cases to rearrangements of the conformational ensembles. [Figure not available: see fulltext.]

Gem Corallium rubrum corals are calcitic biomaterials derived from the branched skeletons of Cnidaria marine invertebrates. Until now the structure of chromophores in Corallium rubrum is still under discussion. First attempts to identify pigments in red corals from Marseille revealed the presence of canthaxanthin. In 2008 Raman spectroscopy suggested that pigments are instead due to unsubstituted polyenes. More recently (2012) ECC theory in Raman spectroscopy stated that the spectrum of Corallium rubrum lies in between carotenoids and unsubstituted polyenes spectra. To confirm and complete the experimental and theoretical results, a group of gem quality specimens of colony skeletons of Corallium rubrum was tested. Rough coral samples were before demineralized with EDTA. HPLC combined with UV was used to separate the pure fractions, on which structural characterization was carried out by ESI-MS and 1H NMR spectroscopy. Results have shown that the colouring pigment profile in Corallium rubrum seems more complex than hitherto considered. HPLC/ UV chromatograms, confirmed by ESI-MS spectra, indicate the presence of two structurally modified carotenoids and of traces of astaxanthin and canthaxanthin. Owing to scarsity of material, 1H NMR spectroscopy did not yield the expected results.

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.

Gem Corallium rubrum corals are calcitic biomaterials derived from the branched skeletons of Cnidaria marine invertebrates. Until now the structure of chromophores in Corallium rubrum is still under discussion. First attempts to identify pigments in red corals from Marseille revealed the presence of canthaxanthin. In 2008 Raman spectroscopy suggested that pigments are instead due to unsubstituted polyenes. More recently (2012) ECC theory in Raman spectroscopy stated that the spectrum of Corallium rubrum lies in between carotenoids and unsubstituted polyenes spectra. To confirm and complete the experimental and theoretical results, a group of gem quality specimens of colony skeletons of Corallium rubrum was tested. Rough coral samples were before demineralized with EDTA. HPLC combined with UV was used to separate the pure fractions, on which structural characterization was carried out by ESI-MS and 1H NMR spectroscopy. Results have shown that the colouring pigment profile in Corallium rubrum seems more complex than hitherto considered. HPLC/ UV chromatograms, confirmed by ESI-MS spectra, indicate the presence of two structurally modified carotenoids and of traces of astaxanthin and canthaxanthin. Owing to scarsity of material, 1H NMR spectroscopy did not yield the expected results.

Lipases resistant to inhibition and denaturation by methanol are valuable tools for biotechnological applications, in particular for biofuel production. Microbial lipases have attracted a great deal of interest because of their stability at high concentrations of organic solvents. Burkholderia cepacia lipase (BCL) is tested here for robustness towards methanol in terms of conformational stability and catalytic activity in transesterification assays. This lipase turns out to be even more tolerant than the homologous and better characterized enzyme from Burkholderia glumae. BCL unfolding transition, as monitored by far-UV circular dichroism (CD) and intrinsic fluorescence, displays a Tm above 60°C in the presence of 50% methanol. The protein unfolds at low pH, and the organic solvent affects the nature of the denatured state under acidic conditions. The protein performs well in transesterification assays upon prolonged incubations at high methanol concentrations. BCL is highly tolerant to methanol and displays particularly high conformational stability under conditions employed for transesterification reactions. These features depict BCL as a promising enzyme for biofuel industry.

Electrospray-ionization mass spectrometry (ESI-MS) is a key tool of structural biology, complementing the information delivered by conventional biochemical and biophysical methods. Yet, the mechanism behind the conformational effects in protein ESI-MS is an object of debate. Two parameters - solvent-accessible surface area (As) and apparent gas-phase basicity (GBapp) - are thought to play a role in controlling the extent of protein ionization during ESI-MS experiments. This review focuses on recent experimental and theoretical investigations concerning the influence of these parameters on ESI-MS results and the structural information that can be derived. The available evidence supports a unified model for the ionization mechanism of folded and unfolded proteins. These data indicate that charge-state distribution (CSD) analysis can provide valuable structural information on normally folded, as well as disordered structures.

Polygalactomannans (PGMs) represent an important family of polysaccharides. They are obtained from the endosperm of leguminous plant seeds and are employed in a growing number of industrial applications as thickening agents and rheology modifiers. Each PGM has a typical mannose/galactose (M/G) ratio (from ~1 to ~5), which determines its solubility, viscosity and other physico-chemical properties. Complex polysaccharides are commonly characterized by proton nuclear magnetic resonance (1H-NMR), whereas mass spectrometry (MS) has not yet seen wide application. In this work, a new quantification method for sugar epimers in PGMs is presented, based on the characteristic fragmentation patterns of mannose and galactose in tandem MS (MS/MS) analyses. Standard galactose and mannose have been analyzed and fragmented in negative-ion mode on a hybrid quadrupole/time-of-flight (qTOF) mass spectrometer equipped with a nano electrospray ionization (ESI) source. The MS/MS spectra indicate accumulation of the same fragments for the two monosaccharides, but significant and reproducible differences in the relative intensities of the product ions. Known mixtures of mannose and galactose have been analyzed by the same procedure to test the applicability of the method for quantification purposes. The resulting peak intensities over the entire MS/MS spectrum can be deconvoluted as a linear combination of the signals from pure mannose and galactose standards, obtaining reliable and reproducible quantification of the epimers. The method has been applied to the characterization of hydrolysis products of PGMs from different species of leguminous plants, such as guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and tara (Caesalpinia spinosa), in order to assess specific susceptibility to hydrolysis conditions.

Short peptides can be designed in silico and synthesized through automated techniques, making them advantageous and versatile protein binders. A number of docking-based algorithms allow for a computational screening of peptides as binders. Here we developed ex-novo peptides targeting the maltose site of the Maltose Binding Protein, the prototypical system for the study of protein ligand recognition.We used a Monte Carlo based protocol, to computationally evolve a set of octapeptides starting from a polialanine sequence. We screened in silico the candidate peptides and characterized their binding abilities by surface plasmon resonance, fluorescence and electrospray ionization mass spectrometry assays. These experiments showed the designed binders to recognize their target with micromolar affinity. We finally discuss the obtained results in the light of further improvement in the exnovo optimization of peptide based binders. Copyright

Short peptides can be designed in silico and synthesized through automated techniques, making them advantageous and versatile protein binders. A number of docking-based algorithms allow for a computational screening of peptides as binders. Here we developed ex-novo peptides targeting the maltose site of the Maltose Binding Protein, the prototypical system for the study of protein ligand recognition.We used a Monte Carlo based protocol, to computationally evolve a set of octapeptides starting from a polialanine sequence. We screened in silico the candidate peptides and characterized their binding abilities by surface plasmon resonance, fluorescence and electrospray ionization mass spectrometry assays. These experiments showed the designed binders to recognize their target with micromolar affinity. We finally discuss the obtained results in the light of further improvement in the exnovo optimization of peptide based binders. Copyright

Polygalactomannans (PGMs) represent an important family of polysaccharides. They are obtained from the endosperm of leguminous plant seeds and are employed in a growing number of industrial applications as thickening agents and rheology modifiers. Each PGM has a typical mannose/galactose (M/G) ratio (from ~1 to ~5), which determines its solubility, viscosity and other physico-chemical properties. Complex polysaccharides are commonly characterized by proton nuclear magnetic resonance (1H-NMR), whereas mass spectrometry (MS) has not yet seen wide application. In this work, a new quantification method for sugar epimers in PGMs is presented, based on the characteristic fragmentation patterns of mannose and galactose in tandem MS (MS/MS) analyses. Standard galactose and mannose have been analyzed and fragmented in negative-ion mode on a hybrid quadrupole/time-of-flight (qTOF) mass spectrometer equipped with a nano electrospray ionization (ESI) source. The MS/MS spectra indicate accumulation of the same fragments for the two monosaccharides, but significant and reproducible differences in the relative intensities of the product ions. Known mixtures of mannose and galactose have been analyzed by the same procedure to test the applicability of the method for quantification purposes. The resulting peak intensities over the entire MS/MS spectrum can be deconvoluted as a linear combination of the signals from pure mannose and galactose standards, obtaining reliable and reproducible quantification of the epimers. The method has been applied to the characterization of hydrolysis products of PGMs from different species of leguminous plants, such as guar (Cyamopsis tetragonolobus), sesbania (Sesbania bispinosa) and tara (Caesalpinia spinosa), in order to assess specific susceptibility to hydrolysis conditions.

Human liver fatty acid binding protein (hL-FABP) is the most abundant cytosolic protein in the liver. This protein plays important roles associated to partitioning of fatty acids (FAs) to specific metabolic pathways, nuclear signaling and protection against oxidative damage. The protein displays promiscuous binding properties and can bind two internal ligands, unlike FABPs from other tissues. Different topologies for the ligand located in the more accessible site have been reported, with either a 'head-in' or 'head-out' orientation of the carboxylate end. Electrospray-ionization mass spectrometry and nuclear magnetic resonance titrations are employed here in order to investigate in further detail the binding properties of this system, the equilibria established in solution and the pH dependence of the complexes. The results are consistent with two binding sites with different affinity and a unique head-out topology for the second molecule of either ligand. Competition experiments indicate a higher affinity for oleic acid relative to palmitic acid at each binding site.

LptA is a periplasmic protein involved in the transport of lipopolysaccharide (LPS) from the inner membrane (IM) to the outer membrane (OM) of Gram-negative bacteria. Growing evidence supports a model in which LptA assembles into oligomers, forming a physical bridge connecting IM and OM. This work investigates LptA oligomer assembly and architecture. Circular dichroism and “native” electrospray-ionization ion-mobility mass spectrometry (ESI-IM-MS) are employed to test concentration dependence of LptA structural features and to analyze the morphology of higher-order aggregates. The results show that LptA progressively assembles into rod-like oligomers without fixed stoichiometry, and grows by an n+1 mechanism up to at least the pentamer. The oligomerization process induces disorder-to-order transitions in the polypeptide chain. Comparison with crystallographic and computational data suggests that these conformational changes likely involve short disordered regions at the N- and C-termini of monomeric LptA. The protein response to thermal denaturation displays strong concentration dependence, indicating that oligomerization increases protein stability. LptA conformational stability can also be enhanced by in vitro LPS binding. The genesis of these fibrillar structures could be relevant for the correct transport of LPS across the bacterial periplasm

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. © 2013 Springer-Verlag Berlin Heidelberg.

This work investigates the effect of chain length on the degree of compaction of intrinsically disordered proteins (IDPs). The three main IDP types, native coil (NC), pre-molten globule (PMG) and molten globule (MG), are compared by means of a compaction index (CI) normalized for chain length. The results point out a strong variability of compactness as a function of chain length within each group, with larger proteins populating more compact states. While qualitative sequence features are responsible for the main differences among groups, chain length seems to have an unspecific effect modulating the extent of compaction within each group. The results are consistent with a cooperative character of the weak interactions responsible for chain collapse.

Aggregation of human ataxin-3 (AT3) into amyloid fibrils is responsible for spinocerebellar ataxia type 3. This protein consists of a folded N-terminal domain (Josephin domain, residues 1-182), a central flexible region (residues 183-291), a poly-glutamine sequence of variable length and a short C-terminal flexible region. Very little is known about the influence of the central flexible region on the conformational and aggregation properties of this protein. The present study aimed to investigate the specific role of this portion of the protein (residues 183-291). Accordingly, protein fragments 1-182 (AT3/182) and 1-291 (AT3/291) were produced and compared by thioflavin-T fluorescence, Fourier transform infrared spectroscopy, CD, intrinsic fluorescence and ESI-MS. It is shown that the central flexible region enhances protein aggregation and can populate conformational states with different degrees of compactness. Both monomeric and dimeric partially-folded forms are identified for both protein fragments under denaturing conditions. Partially-folded monomers and dimers accumulate to a larger extent in AT3/291. These species represent good candidates for early intermediates of the aggregation process under the experimental conditions employed in the present study.

Proteins hosting main β-sheets adopt specific strategies to avoid intermolecular interactions leading to aggregation and amyloid deposition. Human beta-2 microglobulin (β2m) displays a typical immunoglobulin fold and is known to be amyloidogenic in vivo. Upon severe kidney deficiency, β2m accumulates in the bloodstream, triggering, over the years, pathological deposition of large amyloid aggregates in joints and bones. A β-bulge observed on the edge D β-strand of some β2m crystal structures has been suggested to be crucial in protecting the protein from amyloid aggregation. Conversely, a straight D-strand, observed in different crystal structures of monomeric β2m, could promote amyloid aggregation. More recently, the different conformations observed for the β2m D-strand have been interpreted as the result of intrinsic flexibility, rather than being assigned to a functional protective role against aggregation. To shed light on such contrasting picture, the mutation Asp53→Pro was engineered in β2m, aiming to impair the formation of a regular/straight D-strand. Such a mutant was characterized structurally and biophysically by CD, X-ray crystallography and MS, in addition to an assessment of its amyloid aggregation trends in vitro. The results reported in the present study highlight the conformational plasticity of the edge D-strand, and show that even perturbing the D-strand structure through a Pro residue has only marginal effects on protecting β2m from amyloid aggregation in vitro.

Under particular conditions proteins spontaneously aggregate into well-ordered structures called amyloid fibrils. The full elucidation of the fibrillation process requires the identification by biophysical techniques of the conformational and oligomeric species populated during the aggregation mechanism.

The highly dynamic and heterogeneous molecular ensembles characterizing intrinsically disordered proteins (IDP) in solution pose major challenges to the conventional methods for structural analysis. Electrospray ionization-mass spectrometry (ESI-MS) allows direct detection of distinct conformational components, effectively capturing also partially folded and short-lived states. We report the description of two complementary fragments (1-186 and 187-284) of the IDP Sic1, a cyclin-dependent protein kinase inhibitor of yeast Saccharomyces cerevisiae. Structural heterogeneity is noted in both cases, but the two fragments reveal slightly different conformational properties. The results are consistent with previously reported differences between the two protein moieties and corroborate the feasibility of IDP conformational analysis by ESI-MS.

Electrospray-ionization mass spectrometry has recently developed into a central tool of structural biology, which allows the investigation of the structure and the dynamics of protein conformations and protein assemblies. This contribution describes the principles of the main experimental approaches that have been applied to the study of intrinsically disordered proteins and describes some recent example from the literature. In particular, we distinguish between methods that are based on maintenance of non-covalent interactions under electrospray conditions, such as charge-state distribution analysis and ion mobility, from methods based on isotope exchange, in which the protein can be denatured or even digested before mass-spectrometry analysis. All together, these methods offer useful structural information, which is complementary to that gained by other biophysical techniques. Particularly interesting is the possibility to directly monitor distinct conformers in complex mixtures, free from averaging over the molecular population. Although this is a very young field, the examples discussed in this chapter illustrate the contribution that mass-spectrometry methods can give to the structural characterization of intrinsically disordered proteins in the absence and in the presence of interactors

Since the early 1990s, electrospray-ionization mass spectrometry (ESI-MS) has encountered growing interest as a complementary tool to established biochemical and biophysical methods for investigating protein structure and conformation. Nowadays, applications of ESI-MS to protein investigation span from the area of analytical biochemistry to that of structural biology. This review focuses on applications of this technique to the analysis of protein conformational properties and molecular interactions, underscoring their possible relevance for molecular biotechnology, although representing a still very young field. An introductive section presents the major issues related to theoretical and technical aspects of ESI-MS under non-denaturing conditions. Examples from our work and from the literature illustrate which kind of information can be obtained concerning key issues in biotechnology such as stability and aggregation of proteins under both near-native and challenging conditions, and interactions with other proteins, ligands and cofactors