MOSBRI Publications

(Methyl 4-(diphenylphosphoryl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylate-1-yl)oxydanyl was obtained as a key intermediate of the reaction starting from 3,4-dibromo-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxydanyl or (methyl 2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylate-1-yl)oxidanyl. This key compound could be converted into an azido-specific Staudinger ligation-inducing spin label, amino- and thiol-specific spin label, or MITO-CP-like antiproliferative agent.

This PhD thesis describes the structural, biophysical, and functional characterizations of proteins involved in siderophore-mediated iron uptake in Erwinia amylovora, the causal agent of fire blight in apple and pear.

South Tyrol, with its largest apple orchards (18,538 hectares) in Europe, contributes to up to 50% of Italy’s apple production, 15% of Europe’s, and 2% of the world’s apple production. One of the major challenges in apple cultivation is the bacterial infestation in the pre-harvest stage. This problem becomes particularly severe when the pathogen has a history of causing outbreaks and lacks effective chemical control measures. E. amylovora, a destructive pathogen, fits this category and poses a potential threat to apple production. As the use of antibiotics is forbidden in Italy and the current protocols of chemical controls are not efficient, it is crucial to develop novel chemical control against fire blight. For this, studying virulence-related proteins at the structural level is essential. E. amylovora primarily relies on three pathogenicity factors; amylovoran, the Type III secretion system (T3SS), and siderophore-mediated iron uptake (Chapter 1)

This work primarily focuses on the siderophore iron uptake pathway, selecting two target proteins, ViuB (the oxidoreductase) and FhuD (periplasmic binding protein), which are exclusively present in Erwinia species infecting rosaceous plants. The methods for obtaining ViuB, from expression to crystallization and subcloning of FhuD are comprehensively described in (Chapter 2).

ViuB, being a soluble cytoplasmatic protein, was easier to purify. However, crystallization posed a significant challenge. After numerous unsuccessful crystallization trials at UNIBZ, biophysical characterizations and crystallization trials for ViuB was performed at three different laboratories across Europe; BIOCEV, Prague, Robotein at University of Liege, and EMBL in Hamburg (Chapter 3).

Additionally, nanobodies targeting the full-length ViuB protein were produced to stabilize ViuB by binding to its epitopes present in the disordered regions. (Chapter 4).

After several optimizations, particularly adjusting the crystallization temperature and removing tags, ViuB crystals were formed using the Classic and Morpheus screens. Notably, crystals only grew at 4°C. Attempts to incubate ViuB without the His-tag at 20°C with the same batch of protein did not result in crystal formation. The ViuB structure was solved by Small-angle X-ray scattering (SAXS) and X-Ray crystallography at 2.3 Å (Chapter 5).

For another target, the periplasmic binding protein FhuD, soluble protein was not obtained when cloned in vector pMCSG49 and expressed in the cytoplasm of E. coli cells, leading to its subcloning into two EMBL vectors (pETM-41 and pETM-50). pETM-50 encodes His-tagged DsbA, which leads to periplasmic expression, whereas pETM-41 houses a His-tagged MBP solubility tag, increasing solubility in the cytoplasm. Given the crucial role of FhuD in iron uptake, modeling and bioinformatics analysis were performed with its homologs and orthologs proteins to compare the ligand binding pockets (Chapter 6).

Overall, the PhD work adhered strictly to the initial plan, successfully solving the structure of ViuB by X-Ray crystallography. The structure could aid in designing the
inhibitors to disrupt bacterial iron utilization, providing a foundation for developing sustainable chemical controls against fire blight. Determining the structure of ViuB with its substrate bound in the active site and performing the enzymatic assays could be the the future perspective of this work.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a “locked/closed” to an “unlocked/open” state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH· and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an “open” CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the “open” semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Glycerophospholipid membranes are one of the key cellular components. Still, their species-dependent composition and homochirality remain an elusive subject. In the context of the astrophysical circularly polarized light scenario likely involved in the generation of a chiral bias in meteoritic amino and sugar acids in space, and consequently in the origin of life’s homochirality on Earth, this study reports the first measurements of circular dichroism and anisotropy spectra of a selection of glycerophospholipids, their chiral backbones and their analogs. The rather low asymmetry in the interaction of UV/VUV circularly polarized light with sn-glycerol-1/3-phosphate indicates that chiral photons would have been unlikely to directly induce symmetry breaking to membrane lipids. In contrast, the anisotropy spectra of d-3-phosphoglyceric acid and d-glyceraldehyde-3-phosphate unveil up to 20 and 100 times higher maximum anisotropy factor values, respectively. This first experimental report, targeted on investigating the origins of phospholipid symmetry breaking, opens up new avenues of research to explore alternative mechanisms leading to membrane lipid homochirality, while providing important clues for the search for chiral biosignatures of extant and/or extinct life in space, in particular for the ExoMars 2028 mission.

Understanding the impact of the manufacturing environment on therapeutic monoclonal antibody (mAb) structures requires new process analytical technology. Here, we describe the creation of a new reference set for the circular dichroism (CD) spectra of mAbs. Data sets of the highest quality were collected by synchrotron radiation CD for 14 different mAbs in both native and acid-stressed states. Deconvolution of far-UV spectra for the mAb cohort identified two current reference sets (SP175 and SMP180) as assigning accurate secondary structures, irrespective of the analysis program employed. Scrutiny of spectra revealed significant variation in the far-UV and especially near-UV CD of the 14 mAbs. Two spectral features were found to be sensitive to changes in solution pH, i.e., the far-UV positive peak at 201–202 nm and the near-UV negative exciton couplet around 230–240 nm. The latter feature offers attractive possibilities for in-line CD-based monitoring of the mAb structure during manufacture.

Fluorescence-detected linear dichroism (FD-LD) enables one to collect linear dichroism spectra for oriented fluorophores in the presence of other absorbing species and light scattering. The experiment proceeds by scanning the excitation wavelength and using a filter to collect only emitted photons from the fluorophore. Thus, it has the potential to give data with enhanced selectivity and quality. By using a synchrotron radiation light source and fluorescence-detection, we show data for a range of fluorophores in different orienting environments. Film and flow-oriented FD-LD spectra were collected down to 170 nm. Even for flow-oriented liposomes, we have data collected down to 210 nm. For strongly scattering samples, for example, liposomes, FD-LD has the clear advantage that scattering is absent for the longer wavelength fluorescence photons. The collimated and smaller beam size of the synchrotron radiation also gives rise to sharper and more well-defined features in the spectra.

The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), which represents 50% of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS) as well as rare cases of motor and sensory polyneuropathy. We found that high P0ct concentrations affected the membrane properties of bicelles and induced a lamellar-to-inverted hexagonal phase transition, which caused bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semicrystalline lipid domains with potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy also shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterization of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with the successful purification of full-length P0, we have new tools to study the role of P0 in myelin formation and maintenance in vitro.

Hypothesis
Although antimicrobial peptides (AMPs) are a promising class of new antibiotics, their inherent susceptibility to degradation requires nanocarrier-mediated delivery. While cubosome nanocarriers have been extensively studied for delivery of AMPs, we do not currently understand why cubosome encapsulation improves antimicrobial efficacy for some compounds but not others. This study therefore aims to investigate the link between the mechanism of action and permeation efficiency of the peptides, their encapsulation efficacy, and the antimicrobial activity of these systems.

Experiments
Encapsulation and delivery of Indolicidin, and its ultra-short derivative, Priscilicidin, were investigated using SAXS, cryo-TEM and circular dichroism. Molecular dynamics simulations were used to understand the loading of these peptides within cubosomes. The antimicrobial efficacy was assessed against gram-negative (E. coli) and gram-positive (MRSA) bacteria.

Findings
A high ionic strength solution was required to facilitate high loading of the cationic AMPs, with bilayer encapsulation driven by tryptophan and Fmoc moieties. Cubosome encapsulation did not improve the antimicrobial efficacy of the AMPs consistent with their high permeation, as explained by a recent ’diffusion to capture model’. This suggests that cubosome encapsulation may not be an effective strategy for all antimicrobial compounds, paving the way for improved selection of nanocarriers for AMPs, and other antimicrobial compounds.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14’s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Metal-dependent formate dehydrogenases reduce CO₂ with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O₂ exposure.

This work investigates the influence of the enzymatic treatment (Alcalase or trypsin, degree of hydrolysis 5%) and pH (pH 7 or 4) on the interfacial and emulsifying properties of sunflower and olive protein hydrolysates. Independently of the enzymatic treatment, short peptides (1–3 kDa) were the most abundant in sunflower protein hydrolysates, whereas olive protein hydrolysates were richer in large peptides (>10 kDa). Peptides present in all hydrolysates gained in structure when adsorbing at the oil-water interface due to their facial amphiphilicity, with sunflower peptides presenting a more marked β-sheet conformation than olive peptides. Tryptic hydrolysates of both substrates showed higher interfacial adsorption compared to hydrolysates produced with Alcalase, especially at pH 4. All hydrolysates resulted in elastic interfaces, with generally higher values of dilatational complex modulus at pH 7 compared to pH 4. These findings correlated well with the higher emulsifying activity of all hydrolysates at pH 7 than pH 4. Particularly, sunflower protein hydrolysates led to stiffer and more solid-like viscoelastic interfacial layers than olive peptides due to increased interactions between β-sheet peptides at the interface. Indeed, the use of sunflower protein hydrolysates as emulsifiers resulted in 5 wt% oil-in-water emulsions with higher physical stability at both pH 7 and 4 when compared to olive protein hydrolysates.

The main goal of this thesis has been to investigate the interaction of the intrinsically disordered peptide Histatin 5 (Hst5), with phospholipid bilayers, using a mixture of experimental and computational techniques, such as, small- angle X-ray scattering, circular dichroism, neutron reflectometry, quartz-crystal microbalance with dissipation monitoring, atomistic molecular dynamics simulations, and coarse-grained Monte Carlo simulations. Hst5 is of particular interest due to its known antimicrobial effects, where it acts as the first defense against fungal infections in the mouth. With antimicrobial resistance being an increasingly and serious threat against world health, alternative treatments to common antimicrobial agents are needed, where antimicrobial peptides being one option. It is therefore important to understand the mechanism behind their effect, to be able to utilize their properties correctly in pharmaceuticals. In the first study of this thesis, Hst5 was found to spontaneously translocate a phospholipid bilayer, without affecting the structural integrity of the bilayer, thus resulting in a peptide cushion between the supported bilayer and the solid surface. This formation is in line with the antimicrobial effect of Hst5, and has therefore been used as an indication of antimicrobial effect throughout the work conducted in this thesis. The experimental conditions needed for the cushion formation was further determined. In the second study, the role of the amino acid histidine, which is frequently found in Hst5, was investigated to determine its role in the translocation process. The results showed that the penetration depth into the bilayer increases with increasing number of histidines. In this study, it was also suggested that not only the number of histidines, but also their position were important, therefore, a study regarding the order of amino acids was conducted as the ongoing study presented in this thesis. The results indicate a difference in interaction, dependent on the sequence order, however, the underlying explanation is not yet understood. Furthermore, the sequence length of Hst5 was investigated in the third study of this thesis, which only showed small differences at 10 mM NaCl concentration, while in 150 mM NaCl, both the shorter and longer variant display interactions with the bilayer, while Hst5 does not. The final study included in this thesis concerns a different peptide called KEIF, which is the intrinsically disordered N-terminal of magnesium transporter A found in Escherichia coli. This study was conducted with the aim to investigate the hypothesis that surface active intrinsically disordered peptides gain structure upon adsorption, which is related to their function. The peptide became more structured upon adsorption, supporting the presented hypothesis.

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic K_M, k_cat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s^-1 and 2.1 mM, 603 s^-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni²⁺, Cu²⁺, Hg²⁺, and Cd²⁺ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg²⁺ > Cu²⁺ > Cd²⁺ > Ni²⁺. ReAIV is more sensitive to Cu²⁺ and Cd2+, while ReAV is more sensitive to Hg²⁺ and Ni²⁺, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd²⁺ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg²⁺ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl⁻ anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.

Recently, it has been hypothesized that alpha-synuclein protein strain morphology may be associated with clinical subtypes of alpha-synucleinopathies, like Parkinson’s disease and multiple system atrophy. However, direct evidence is lacking due to the caveat of conformation-specific characterization of protein strain morphology. Here we present a new cell model based in vitro method to explore various alpha-synuclein (αsyn) aggregate morphotypes. We performed a spectroscopic investigation of the HEK293 cell model, transfected with human wildtype-αsyn and A53T-αsyn variants, using the amyloid fibril-specific heptameric luminescent oligomeric thiophene h-FTAA. The spectral profile of h-FTAA binding to aggregates displayed a blue-shifted spectrum with a fluorescence decay time longer than in PBS, suggesting a hydrophobic binding site. In vitro spectroscopic binding characterization of h-FTAA with αsyn pre-formed fibrils suggested a binding dissociation constant K_d < 100 nM. The cells expressing the A53T-αsyn and human wildtype-αsyn were exposed to recombinant pre-formed fibrils of human αsyn. The ensuing intracellular aggregates were stained with h-FTAA followed by an evaluation of the spectral features and fluorescence lifetime of intracellular αsyn/h-FTAA, in order to characterize aggregate morphotypes. This study exemplifies the use of cell culture together with conformation-specific ligands to characterize strain morphology by investigating the spectral profiles and fluorescence lifetime of h-FTAA, based upon its binding to a certain αsyn aggregate. This study paves the way for toxicity studies of different αsyn strains in vitro and in vivo. Accurate differentiation of specific alpha-synucleinopathies is still limited to advanced disease stages. However, early subtype-specific diagnosis is of the utmost importance for prognosis and treatment response. The potential association of αsyn aggregates morphotypes detected in biopsies or fluids to disease phenotypes would allow for subtype-specific diagnosis in subclinical disease stage and potentially reveal new subtype-specific treatment targets. Notably, the method may be applied to the entire spectrum of neurodegenerative diseases by using a combination of conformation-specific ligands in a physicochemical environment together with other types of polymorphic amyloid variants and assess the conformation-specific features of various protein pathologies.

The gelation behaviour of two different pea protein isolates and one soy protein isolate were investigated with a focus on the role of the protein properties. Protein solubility was the lowest in pH 3 citrate-phosphate buffer (<10% w/w), increased in pH 7.4 phosphate-buffered saline (12–21% w/w), and was the highest in pH 7.6 MilliQ water (~20–40% w/w). Heat-induced gelation conditions for the protein sources were sensitive to both the soluble and the insoluble fractions as obtained during extraction. At low protein concentrations (≤5% w/v), the proteins started to lose their viscoelastic behaviour and exhibited predominantly viscous properties. Fitting of the fractional Kelvin-Voigt model to the frequency sweeps showed an increase in the fractal gel strength with increasing protein concentration. Secondary structures of the soluble species showed mostly unordered proteins, suggesting that the proteins were denatured during the commercial extraction process although gelation has to date been suggested to be highly dependent on the denaturation of soluble proteins. Synchrotron Radiation Circular Dichroism measurements of the insoluble proteins showed a significant amount of ordered protein structures. SEM imaging of the gels also suggested a new gelation pathway in which insoluble proteins act as dispersed fillers within a continuous matrix of soluble proteins. The goal of this research is to elucidate the the role of different protein fractions, globulins and albumins, and their secondary structure in the formation of a gel network and how this affects their viscoelastic behaviour.

Under specific conditions, some proteins can self-assemble into fibrillar structures called amyloids. Initially, these proteins were associated with neurodegenerative diseases in eucaryotes. Nevertheless, they have now been identified in the three domains of life. In bacteria, they are involved in diverse biological processes and are usually useful for the cell. For this reason, they are classified as “functional amyloids”. In this work, we focus our analysis on a bacterial functional amyloid called Hfq. Hfq is a pleiotropic regulator that mediates several aspects of genetic expression, mainly via the use of small noncoding RNAs. Our previous work showed that Hfq amyloid-fibrils interact with membranes. This interaction influences Hfq amyloid structure formation and stability, but the specifics of the lipid on the dynamics of this process is unknown. Here, we show, using spectroscopic methods, how lipids specifically drive and modulate Hfq amyloid assembly or, conversely, its disassembly. The reported effects are discussed in light of the consequences for bacterial cell life.

Metal-dependent formate dehydrogenases are very promising targets for enzyme optimization and design of bio-inspired catalysts for CO₂ reduction, towards innovative strategies for climate change mitigation. For effective application of these enzymes, the catalytic mechanism must be better understood, and the molecular determinants clarified. Despite numerous studies, several doubts persist, namely regarding the role played by the possible dissociation of the SeCys ligand from the Mo/W active site. Additionally, the oxygen sensitivity of these enzymes must also be understood as it poses an important obstacle for biotechnological applications. This work presents a combined biochemical, spectroscopic, and structural characterization of Desulfovibrio vulgaris FdhAB (DvFdhAB) when exposed to oxygen in the presence of a substrate (formate or CO₂). This study reveals that O₂ inactivation is promoted by the presence of either substrate and involves forming a different species in the active site, captured in the crystal
structures, where the SeCys ligand is displaced from tungsten coordination and replaced by a dioxygen or peroxide molecule. This form was reproducibly obtained and supports the conclusion that, although W-DvFdhAB can catalyse the oxidation of formate in the presence of oxygen for some minutes, it gets irreversibly inactivated after prolonged O₂ exposure in the presence of either substrate.

Dynamic light scattering (DLS) is routinely employed to assess the homogeneity and size-distribution profile of samples containing microscopic particles in suspension or solubilized polymers. In this work, Raynals, user-friendly software for the analysis of single-angle DLS data that uses the Tikhonov–Phillips regularization, is introduced. Its performance is evaluated on simulated and experimental data generated by different DLS instruments for several proteins and gold nanoparticles. DLS data can easily be misinterpreted and the simulation tools available in Raynals allow the limitations of the measurement and its resolution to be understood. It was designed as a tool to address the quality control of biological samples during sample preparation and optimization and it helps in the detection of aggregates, showing the influence of large particles. Lastly, Raynals provides flexibility in the way that the data are presented, allows the export of publication-quality figures, is free for academic use and can be accessed online on the eSPC data-analysis platform at https://spc.embl-hamburg.de/.

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab, using some functions of Easyspin. An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (g(y), g(z)) and others are determined (A(z), g(x)) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters.

In a previous study, the coexistence of different aggregation pathways of insulin and beta-amyloid (A beta) peptides was demonstrated by correlative stimulated emission depletion (STED) microscopy and atomic force microscopy (AFM). This had been explained by suboptimal proteins labeling strategies that generate heterogeneous populations of aggregating species. However, because of the limited number of proteins considered, the failure of the fluorescent labeling that occurs in a large portion of the aggregating fibrils observed for insulin and A beta peptides, could not be considered a general phenomenon valid for all molecular systems. Here, we investigated the aggregation process of alpha-synuclein (alpha-syn), an amyloidogenic peptide involved in Parkinson’s disease, which is significantly larger (MW similar to 14 kDa) than insulin and A beta, previously investigated. The results showed that an unspecific labeling procedure, such as that previously adopted for shorter proteins, reproduced the coexistence of labeled/unlabeled fibers. Therefore, a site-specific labeling method was developed to target a domain of the peptide scarcely involved in the aggregation process. Correlative STED-AFM illustrated that all fibrillar aggregates derived from the aggregation of alpha-syn at the dye-to-protein ratio of 1 : 22 were fluorescent. These results, demonstrated here for the specific case of alpha-syn, highlight that the labeling artifacts can be avoided by careful designing the labeling strategy for the molecular system under investigation. The use of a label-free correlative microscopy technique would play a crucial role in the control of the setting of these conditions.

Metal-based formate dehydrogenases are molybdenum or tungsten-dependent enzymes that catalyze the interconversion between formate and CO2. According to the current consensus, the metal ion of the catalytic center in its active form is coordinated by 6S (or 5S and 1 Se) atoms, leaving no free coordination sites to which formate could bind to the metal. Some authors have proposed that one of the active site ligands decoordinates during turnover to allow formate binding. Another proposal is that the oxidation of formate takes place in the second coordination sphere of the metal. Here, we have used electrochemical steady-state kinetics to elucidate the order of the steps in the catalytic cycle of two formate dehydrogenases. Our results strongly support the “second coordination sphere” hypothesis.

Biomimetic cubic phases can be used for protein encapsulation in a variety of applications such as biosensors and drug delivery. Cubic phases with a high concentration of cholesterol and phospholipids were obtained herein. It is shown that the cubic phase structure can be maintained with a higher concentration of biomimetic membrane additives than has been reported previously. Opposing effects on the curvature of the membrane were observed upon the addition of phospholipids and cholesterol. Furthermore, the coronavirus fusion peptide significantly increased the negative curvature of the biomimetic membrane with cholesterol. We show that the viral fusion peptide can undergo structural changes leading to the formation of hydrophobic alpha-helices that insert into the lipid bilayer. This is of high importance, as a fusion peptide that induces increased negative curvature as shown by the formation of inverse hexagonal phases allows for greater contact area between two membranes, which is required for viral fusion to occur. The cytotoxicity assay showed that the toxicity toward HeLa cells was dramatically decreased when the cholesterol or peptide level in the nanoparticles increased. This suggests that the addition of cholesterol can improve the biocompatibility of the cubic phase nanoparticles, making them safer for use in biomedical applications. As the results, this work improves the potential for the biomedical end-use applications of the nonlamellar lipid nanoparticles and shows the need of systematic formulation studies due to the complex interplay of all components.

UreG is a cytosolic GTPase involved in the maturation network of urease, an Ni-containing bacterial enzyme. Previous investigations in vitro showed that UreG features a flexible tertiary organization, making this protein the first enzyme discovered to be intrinsically disordered. To determine whether this heterogeneous behavior is maintained in the protein natural environment, UreG structural dynamics was investigated directly in intact bacteria by in-cell EPR. This approach, based on site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy, enables the study of proteins in their native environment. The results show that UreG maintains heterogeneous structural landscape in-cell, existing in a conformational ensemble of two major conformers, showing either random coil-like or compact properties. These data support the physiological relevance of the intrinsically disordered nature of UreG and indicates a role of protein flexibility for this specific enzyme, possibly related to the regulation of promiscuous protein interactions for metal ion delivery.

Antimicrobial resistance poses a major threat to public health. Given the paucity of novel antimicrobials to treat resistant infections, the emergence of multidrug-resistant bacteria renewed interest in antimicrobial peptides as potential therapeutics. This study designed a new analog of the antimicrobial peptide Plantaricin 149 (Pln149-PEP20) based on previous Fmoc-peptides. The minimal inhibitory concentrations of Pln149-PEP20 were determined for 60 bacteria of different species and resistance profiles, ranging from 1 mg/L to 128 mg/L for Gram-positive bacteria and 16 to 512 mg/L for Gram-negative. Furthermore, Pln149-PEP20 demonstrated excellent bactericidal activity within one hour. To determine the propensity to develop resistance to Pln149-PEP20, a directed-evolution in vitro experiment was performed. Whole-genome sequencing of selected mutants with increased MICs and wild-type isolates revealed that most mutations were concentrated in genes associated with membrane metabolism, indicating the most likely target of Pln149-PEP20. Synchrotron radiation circular dichroism showed how this molecule disturbs the membranes, suggesting a carpet mode of interaction. Membrane depolarization and transmission electron microscopy assays supported these two hypotheses, although a secondary intracellular mechanism of action is possible. The molecule studied in this research has the potential to be used as a novel antimicrobial therapy, although further modifications and optimization remain possible.

Bunyaviruses constitute a large and diverse group of viruses encompassing many emerging pathogens, such as Rift Valley fever virus (family Phenuiviridae), with public and veterinary health relevance but with very limited medical countermeasures are available. For the development of antiviral strategies, the identification and validation of virus-specific targets would be of high value. The cap-snatching mechanism is an essential process in the life cycle of bunyaviruses to produce capped mRNAs, which are then recognized and translated into viral proteins by the host cell translation machinery. Cap-snatching involves cap-binding as well as endonuclease functions and both activities have been demonstrated to be druggable in related influenza viruses. Here, we explore the suitability of the phenuivirus cap-binding function as a target in medium- and high-throughput drug discovery approaches. We developed a range of in vitro assays aiming to detect the interaction between the cap-binding domain (CBD) and the analogue of its natural cap-ligand m7GTP. However, constricted by its shallow binding pocket and low affinity for m7GTP, we conclude that the CBD has limited small molecule targeting potential using classical in vitro drug discovery approaches.

Bacteria can form organized community called biofilm allowing them to colonize the surfaces and to resist antimicrobial treatments and host defenses. The second messenger 3’-5’cyclic diguanylic acid (c-di-GMP) controls biofilm formation, maintenance, and dispersion in response to environmental signals, including nutrients and stressors. The intracellular levels of c-di-GMP are controlled by the rate of its synthesis and degradation, regulated by diguanylate cyclases (GGDEF signature) and phosphodiesterase (EAL and HD- GYP signatures), respectively. Among nutrients, Arginine represents one key metabolite in biofilm formation being at the crossroad of many metabolic processes. Moreover, in Pseudomonas aeruginosa, Arginine is the substrate for ATP production under low O2 levels and plays a key role in chronic infections, biofilm and antibiotic resistance. RmcA (Redox modulator of c-di-GMP) is a multidomain phosphodiesterase that links redox conditions to colony morphogenesis in P. aeruginosa, by modulating levels of c-di-GMP and wrinkling phenotype in response to phenazine availability. RmcA is a membrane protein composed by a periplasmic VFT domain, a transmembrane helix, four Per-Arnt-Sim (PAS) domains that are responsible for the periplasmic signal transduction to the catalytic moiety (GGDEF-EAL). RmcA is involved in biofilm maintenance pathway of P. aeruginosa, and the characterization of this protein is important to gain details of the molecular mechanism in terms of electron perceiving and nutrient sensing to finally control c-di-GMP consumption activity and treat chronic infection. Moreover, understanding mechanism involved in biofilm maintenance is relevant also in biotechnology field. In this thesis we have contributed to gain mechanistic details on the RmcA-based signal transduction by reaching three main goals: 1) The characterization of cytosolic portion of RmcA (cRmcA). I have found that RmcA regulates its phosphodiesterase activity by detecting the intracellular redox potential FAD/ FADH2 via the PASd domain. 2) The RmcA control via VFT domain in response to arginine. I have isolated the membrane protein and in vitro obtained the transduction of the periplasmic signal to the catalytic portion, resulting in the regulation of the phosphodiesterase activity in response to arginine. 3) The effect of RmcA in controlling P. putida metabolism in response to arginine. I have developed a novel approach for measuring the bacterial energy metabolism; the ΔrmcA mutant is affected by Arginine when it is the only carbon source.

The ability of many bacteria to form biofilms contributes to their resilience and makes infections more difficult to treat. Biofilm growth leads to the formation of internal oxygen gradients, creating hypoxic subzones where cellular reducing power accumulates, and metabolic activities can be limited. The pathogen Pseudomonas aeruginosa counteracts the redox imbalance in the hypoxic biofilm subzones by producing redox-active electron shuttles (phenazines) and by secreting extracellular matrix, leading to an increased surface area-to-volume ratio, which favors gas exchange. Matrix production is regulated by the second messenger bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) in response to different environmental cues. RmcA (Redox modulator of c-di-GMP) from P. aeruginosa is a multidomain phosphodiesterase (PDE) that modulates c-di-GMP levels in response to phenazine availability. RmcA can also sense the fermentable carbon source arginine via a periplasmic domain, which is linked via a transmembrane domain to four cytoplasmic Per-Arnt-Sim (PAS) domains followed by a diguanylate cyclase (DGC) and a PDE domain. The biochemical characterization of the cytoplasmic portion of RmcA reported in this work shows that the PAS domain adjacent to the catalytic domain tunes RmcA PDE activity in a redox-dependent manner, by differentially controlling protein conformation in response to FAD or FADH2. This redox-dependent mechanism likely links the redox state of phenazines (via FAD/FADH2 ratio) to matrix production as indicated by a hyperwrinkling phenotype in a macrocolony biofilm assay. This study provides insights into the role of RmcA in transducing cellular redox information into a structural response of the biofilm at the population level. Conditions of resource (i.e. oxygen and nutrient) limitation arise during chronic infection, affecting the cellular redox state and promoting antibiotic tolerance. An understanding of the molecular linkages between condition sensing and biofilm structure is therefore of crucial importance from both biological and engineering standpoints.

Amino acids are crucial in nitrogen cycling and to shape the metabolism of microorganisms. Among them, arginine is a versatile molecule able to sustain nitrogen, carbon, and even ATP supply and to regulate multicellular behaviors such as biofilm formation. Arginine modulates the intracellular levels of 3’–5′ cyclic diguanylic acid (c-di-GMP), a second messenger that controls biofilm formation, maintenance and dispersion. In Pseudomonas putida, KT2440, a versatile microorganism with wide biotechnological applications, modulation of c-di-GMP levels by arginine requires the transcriptional regulator ArgR, but the connections between arginine metabolism and c-di-GMP are not fully characterized. It has been recently demonstrated that arginine can be perceived by the opportunistic human pathogen Pseudomonas aeruginosa through the transducer RmcA protein (Redox regulator of c-di-GMP), which can directly decrease c-di-GMP levels and possibly affect biofilm architecture. A RmcA homolog is present in P. putida, but its function and involvement in arginine perceiving or biofilm life cycle had not been studied. Here, we present a preliminary characterization of the RmcA-dependent response to arginine in P. putida in modulating biofilm formation, c-di-GMP levels, and energy metabolism. This work contributes to further understanding the molecular mechanisms linking biofilm homeostasis and environmental adaptation.

Histatin 5 is a histidine-rich, intrinsically disordered, multifunctional saliva protein known to act as a first line of defense against oral candidiasis caused by Candida albicans. An earlier study showed that, upon interaction with a common model bilayer, a protein cushion spontaneously forms underneath the bilayer. Our hypothesisis that this effect is of electrostatic origin and that the observed behavior is due to proton charge fluctuations of the histidines, promoting attractive electrostatic interactions between the positively charged proteins and the anionic surfaces, with concomitant counterion release. Here we are investigating the role of the histidines in more detail by defining a library of variants of the peptide, where the former have been replaced by the pH-insensitive amino acid glutamine. By using experimental techniques such as circular dichroism, small angle X-ray scattering, quartz crystal microbalance with dissipation monitoring, and neutron reflectometry, it was determined that changing the number of histidines in the peptide sequence did not affect the structure of the peptide dissolved in solution. However, it was shown to affect the penetration depth of the peptide into the bilayer, where all variants except the one with zero histidines were found below the bilayer. A decrease in the number of histidine from the original seven to zero decreases the ability of the peptide to penetrate the bilayer, and the peptide is then also found residing within the bilayer. We hypothesize that this is due to the ability of the histidines to charge titrate, which charges up the peptide, and enables it to penetrate and translocate through the lipid bilayer.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral polyneuropathy in humans, and its subtypes are linked to mutations in dozens of different genes, including the gene coding for ganglioside-induced differentiation-associated protein 1 (GDAP1). The main GDAP1-linked CMT subtypes are the demyelinating CMT4A and the axonal CMT2K. Over a hundred different missense CMT mutations in the GDAP1 gene have been reported. However, despite implications for mitochondrial fission and fusion, cytoskeletal interactions, and response to reactive oxygen species, the etiology of GDAP1-linked CMT is poorly understood at the protein level. Based on earlier structural data, CMT-linked mutations could affect intramolecular interaction networks within the GDAP1 protein. We carried out structural and biophysical analyses on several CMT-linked GDAP1 protein variants and describe new crystal structures of the autosomal recessive R120Q and the autosomal dominant A247V and R282H GDAP1 variants. These mutations reside in the structurally central helices ⍺3, ⍺7, and ⍺8. In addition, solution properties of the CMT mutants R161H, H256R, R310Q, and R310W were analysed. All disease variant proteins retain close to normal structure and solution behaviour. All mutations, apart from those affecting Arg310 outside the folded GDAP1 core domain, decreased thermal stability. In addition, a bioinformatics analysis was carried out to shed light on the conservation and evolution of GDAP1, which is an outlier member of the GST superfamily. GDAP1-like proteins branched early from the larger group of GSTs. Phylogenetic calculations could not resolve the exact early chronology, but the evolution of GDAP1 is roughly as old as the splits of archaea from other kingdoms. Many known CMT mutation sites involve conserved residues or interact with them. A central role for the ⍺6-⍺7 loop, within a conserved interaction network, is identified for GDAP1 protein stability. To conclude, we have expanded the structural analysis on GDAP1, strengthening the hypothesis that alterations in conserved intramolecular interactions may alter GDAP1 stability and function, eventually leading to mitochondrial dysfunction, impaired protein-protein interactions, and neuronal degeneration.

The mitochondrial outer membrane creates a diffusion barrier between the cytosol and the mitochondrial intermembrane space, allowing the exchange of metabolic products, important for efficient mitochondrial function in neurons. The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial outer membrane protein with a critical role in mitochondrial dynamics and metabolic balance in neurons. Missense mutations in the GDAP1 gene are linked to the most common human peripheral neuropathy, Charcot-Marie-Tooth disease (CMT). GDAP1 is a distant member of the glutathione-S-transferase (GST) superfamily, with unknown enzymatic properties or functions at the molecular level. The structure of the cytosol-facing GST-like domain has been described, but there is no consensus on how the protein interacts with the mitochondrial outer membrane. Here, we describe a model for GDAP1 assembly on the membrane using peptides vicinal to the GDAP1 transmembrane domain. We used oriented circular dichroism spectroscopy (OCD) with synchrotron radiation to study the secondary structure and orientation of GDAP1 segments at the outer and inner surfaces of the outer mitochondrial membrane. These experiments were complemented by small-angle X-ray scattering, providing the first experimental structural models for full-length human GDAP1. The results indicate that GDAP1 is bound into the membrane via a single transmembrane helix, flanked by two peripheral helices interacting with the outer and inner leaflets of the mitochondrial outer membrane in different orientations. Impairment of these interactions could be a mechanism for CMT in the case of missense mutations affecting these segments instead of the GST-like domain.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.

In prokaryotic organisms, lower eukaryotes and plants, some important biological reactions are catalyzed by nickel-dependent enzymes, making this metal ion essential microelement for their life. On the other hand, excessive concentration of nickel into the cell, or prolonged exposure to nickel compounds, has toxic effects in living organisms. In addition, nickel has been classified by IARC as Group I human carcinogen, because of the correlation between its inhalation and increased incidence of nasal and lung cancers. The aim of this work was to investigate the nickel impact on human health, considering both its direct role on human cells and its indirect effect as essential element for human important bacteria. In humans, nickel induces N-myc downstream regulated gene 1 (NDRG1) expression, recently proposed as new target in cancer therapy. CD, light scattering and ITC were applied on the recombinant full-length protein and its C-terminal intrinsically disordered domain, for studying the NDRG1 structural and functional properties. In particular, the fold and dynamics of the C-terminal region were examined by NMR spectroscopy and site-directed spin labeling coupled to EPR, showing the features of an intrinsically disordered region. In nickel-dependent bacteria, nickel metabolism is strictly regulated, through the activity of different transcription factors. In Streptomyces griseus the expression of two superoxide dismutases (SODs) is antagonistically regulated by nickel thanks to the transcriptional complex SgSrnR/SgSrnQ. The SgSrnR protein was heterologously expressed and its activity as possible nickel sensor studied. DNaseI footprinting and β-galactosidase gene reporter assays revealed that SgSrnR functions as transcriptional activator, prompting the hypothesis of a new model to describe the activity of this complex. In addition, ITC, NMR and X-ray crystallography demonstrated that SgSrnR presents the fold typical of ArsR/SmtB transcription factors and low metal binding affinity, non compatible with a role as a nickel-sensor, function probably played by its partner SgSrnQ.

This study investigates possible structural changes of an intrinsically disordered protein (IDP) when it adsorbs to a solid surface. Experiments on IDPs primarily result in ensemble averages due to their high dynamics. Therefore, molecular dynamics (MD) simulations are crucial for obtaining more detailed information on the atomistic and molecular levels. An evaluation of seven different force field and water model combinations have been applied: ( A) CHARMM36IDPSFF + CHARMM-modified TIP3P, (B) CHARMM36IDPSFF + TIP4P-D, (C) CHARMM36m + CHARMM-modified TIP3P, (D) AMBER99SB-ILDN + TIP3P, (E) AMBER99SB-ILDN + TIP4P-D, (F) AMBERff03ws + TIP4P/2005, and (G) AMBER99SB-disp + disp-water. The results have been qualitatively compared with those of small-angle X-ray scattering, synchrotron radiation circular dichroism spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. The model IDP corresponds to the first 33 amino acids of the N-terminal of the magnesium transporter A (MgtA) and is denoted as KEIF. With a net charge of +3, KEIF is found to adsorb to the anionic synthetic clay mineral Laponite (R) due to the increase in entropy from the concomitant release of counterions from the surface. The experimental results show that the peptide is largely disordered with a random coil conformation, whereas the helical content (alpha- and/or 3(10)-helices) increased upon adsorption. MD simulations corroborate these findings and further reveal an increase in polyproline II helices and an extension of the peptide conformation in the adsorbed state. In addition, the simulations provided atomistic resolution of the adsorbed ensemble of structures, where the arginine residues had a high propensity to form hydrogen bonds with the surface. Simulations B, E, and G showed significantly better agreement with experiments than the other simulations. Particularly noteworthy is the discovery that B and E with TIP4P-D water had superior performance to their corresponding simulations A and D with TIP3P-type water. Thus, this study shows the importance of the water model when simulating IDPs and has also provided an insight into the structural changes of surface-active IDPs induced by adsorption, which may play an important role in their function.

TRP channels sense temperatures ranging from noxious cold to noxious heat. Whether specialized TRP thermosensor modules exist and how they control channel pore gating is unknown. We studied purified human TRPA1 (hTRPA1) truncated proteins to gain insight into the temperature gating of hTRPA1. In patch-clamp bilayer recordings, Delta 1-688 hTRPA1, without the N-terminal ankyrin repeat domain (N-ARD), was more sensitive to cold and heat, whereas Delta 1-854 hTRPA1, also lacking the S1-S4 voltage sensing-like domain (VSLD), gained sensitivity to cold but lost its heat sensitivity. In hTRPA1 intrinsic tryptophan fluorescence studies, cold and heat evoked rearrangement of VSLD and the C-terminus domain distal to the transmembrane pore domain S5-S6 (CTD). In whole-cell electrophysiology experiments, replacement of the CTD located cysteines 1021 and 1025 with alanine modulated hTRPA1 cold responses. It is proposed that hTRPA1 CTD harbors cold and heat sensitive domains allosterically coupled to the S5-S6 pore region and the VSLD, respectively.

Human osteocalcin (OC) undergoes reversible, vitamin K-dependent γ-carboxylation at three glutamic acid residues, modulating its release from bones and its hormonal roles. A complete understanding of OC roles and structure–activity relationships is still lacking, as only uncarboxylated and few differently carboxylated variants have been considered so far. To fill this lack of knowledge, a comprehensive experimental and computational investigation of the structural properties and calcium-binding activity of all the OC variants is reported here. Such a comparative study indicates that the carboxylation sites are not equivalent and differently affect the OC structure and interaction with calcium, properties that are relevant for the modulation of OC functions. This study also discloses cooperative effects and provides structural and mechanistic interpretation. The disclosed peculiar features of each carboxylated proteoform strongly suggest that considering all eight possible OC variants in future studies may help rationalize some of the conflicting hypotheses observed in the literature.

High-risk human papillomaviruses (HPVs) cause various cancers. While type-specific prophylactic vaccines are available, additional anti-viral strategies are highly desirable. Initial HPV cell entry involves receptor-switching induced by structural capsid modifications. These modifications are initiated by interactions with cellular heparan sulphates (HS), however, their molecular nature and functional consequences remain elusive. Combining virological assays with hydrogen/deuterium exchange mass spectrometry, and atomic force microscopy, we investigate the effect of capsid-HS binding and structural activation. We show how HS-induced structural activation requires a minimal HS-chain length and simultaneous engagement of several binding sites by a single HS molecule. This engagement introduces a pincer-like force that stabilizes the capsid in a conformation with extended capsomer linkers. It results in capsid enlargement and softening, thereby likely facilitating L1 proteolytic cleavage and subsequent L2-externalization, as needed for cell entry. Our data supports the further devising of prophylactic strategies against HPV infections.

Antimicrobial resistance is responsible for an alarming number of deaths, estimated at 5 million per year. To combat priority pathogens, like Helicobacter pylori, the development of novel therapies is of utmost importance. Understanding the molecular alterations induced by medications is critical for the design of multi-targeting treatments capable of eradicating the infection and mitigating its pathogenicity. However, the application of bulk omics approaches for unraveling drug molecular mechanisms of action is limited by their inability to discriminate between target-specific modifications and off-target effects. This study introduces a multi-omics method to overcome the existing limitation. For the first time, the Proteome Integral Solubility Alteration (PISA) assay is utilized in bacteria in the PISA-Express format to link proteome solubility with different and potentially immediate responses to drug treatment, enabling us the resolution to understand target-specific modifications and off-target effects. This study introduces a comprehensive method for understanding drug mechanisms and optimizing the development of multi-targeting antimicrobial therapies.

The need to combat antimicrobial resistance is becoming more and more pressing. Here we investigate the working mechanism of a small cationic agent, N-alkylamide 3d, by conventional and high-speed atomic force microscopy. We show that N-alkylamide 3d interacts with the membrane of Staphylococcus aureus, where it changes the organization and dynamics of lipid domains. After this initial step, supramolecular structures of the antimicrobial agent attach on top of the affected membrane gradually, covering it entirely. These results demonstrate that lateral domains in the bacterial membranes might be affected by small antimicrobial agents more often than anticipated. At the same time, we show a new dual-step activity of N-alkylamide 3d that not only destroys the lateral membrane organization but also effectively covers the whole membrane with aggregates. This final step could render the membrane inaccessible from the outside and possibly prevent signaling and waste disposal of living bacteria.

Proteins form the fastest-growing therapeutic class. Due to their intrinsic instability, loss of native structure is common. Structure alteration must be carefully evaluated as structural changes may jeopardize the efficiency and safety of the protein-based drugs. Hydrogen deuterium exchange (HDX) has long been used to evaluate protein structure and dynamics. The rate of exchange constitutes a sensitive marker of the conformational state of the protein and of its stability. It is often monitored by mass spectrometry. Fourier transform infrared (FTIR) spectroscopy is another method with very promising capabilities. Combining protein microarrays with FTIR imaging resulted in high throughput HDX FTIR measurements. BaF₂ slides bearing the protein microarrays were covered by another slide separated by a spacer, allowing us to flush the cell continuously with a flow of N₂ gas saturated with ²H₂O. Exchange occurred simultaneously for all proteins and single images covering ca. 96 spots of proteins that could be recorded on-line at selected time points. Each protein spot contained ca. 5 ng protein, and the entire array covered 2.5 × 2.5 mm². Furthermore, HDX could be monitored in real time, and the experiment was therefore not subject to back-exchange problems. Analysis of HDX curves by inverse Laplace transform and by fitting exponential curves indicated that quantitative comparison of the samples is feasible. The paper also demonstrates how the whole process of analysis can be automatized to yield fast analyses.

The study of how the intracellular medium influences protein structural dynamics and protein−protein interactions is a captivating area of research for scientists aiming to comprehend biomolecules in their native environment. As the cellular environment can hardly be reproduced in vitro, direct investigation of biomolecules within cells has attracted growing interest in the past two decades. Among magnetic resonances, site-directed spin labeling coupled to electron paramagnetic resonance spectroscopy (SDSLEPR) has emerged as a powerful tool for studying the structural properties of biomolecules directly in cells. Since the first in-cell EPR experiment was reported in 2010, substantial progress has been made, and this Review provides a detailed overview of the developments and applications of this spectroscopic technique. The strategies available for preparing a cellular sample and the EPR methods that can be applied to cells will be discussed. The array of spin labels available, along with their strengths and weaknesses in cellular contexts, will also be described. Several examples will illustrate how in-cell EPR can be applied to different biological systems and how the cellular environment affects the structural and dynamic properties of different proteins. Lastly, the Review will focus on the future developments expected to expand the capabilities of this promising technique.

Dynamic processes and structural changes of biological molecules are essential to life. While conventional atomic force microscopy (AFM) is able to visualize molecules and supramolecular assemblies at sub-nanometer resolution, it cannot capture dynamics because of its low imaging rate. The introduction of high-speed atomic force microscopy (HS-AFM) solved this problem by providing a large increase in imaging velocity. Using HS-AFM, one is able to visualize dynamic molecular events with high spatiotem-poral resolution under near-to physiological conditions. This approach opened new windows as finally dynamics of biomolecules at sub-nanometer resolution could be studied. Here we describe the working principles and an operation protocol for HS-AFM imaging and characterization of biological samples in liquid.

Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes. The mechanism of action of the antibacterial tripeptide AMC-109 is unclear. Here, Melcrova et al. show that AMC-109 self-assembles into stable aggregates with a cationic surface, and then individual peptides insert into the bacterial membrane and disrupt membrane nanodomains, thus affecting membrane function without forming pores.

Quality control of drug products is of paramount importance in the pharmaceutical world. It ensures product safety, efficiency, and consistency. In the case of complex biomolecules such as therapeutic proteins, small variations in bioprocess parameters can induce substantial variations in terms of structure, impacting the drug product quality. Conditions for obtaining highly reproducible grafting of 11-mercaptoundecanoic acid were determined. On that basis, we developed an easy-to-use, cost effective, and timesaving biosensor based on ATR-FTIR spectroscopy able to detect immunoglobulins during their production. A germanium crystal, used as an internal reflection element (IRE) for FTIR spectroscopy, was covalently coated with immunoglobulin-binding proteins. This thereby functionalized surface could bind only immunoglobulins present in complex media such as culture media or biopharmaceutical products. The potential subsequent analysis of their structure by ATR-FTIR spectroscopy makes this biosensor a powerful tool to monitor the production of biotherapeutics and assess important critical quality attributes (CQAs) such as high-order structure and aggregation level.

The loss of native structure is common in proteins. Among others, aggregation is one structural modification of particular importance as it is a major concern for the efficiency and safety of biotherapeutic proteins. Yet, recognizing the structural features associated with intermolecular bridging in a high-throughput manner remains a challenge. We combined here the use of protein microarrays spotted at a density of ca 2500 samples per cm(2) and Fourier transform infrared (FTIR) imaging to analyze structural modifications in a set of 85 proteins characterized by widely different secondary structure contents, submitted or not to mild denaturing conditions. Multivariate curve resolution alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subject to denaturing conditions. In the native protein set, 6 components were found to be sufficient to obtain good modeling of the spectra. Furthermore, their shape allowed them to be assigned to alpha-helix, beta-sheet, and other structures. Their content in each protein was correlated with the known secondary structure, confirming these assignments. In the denatured proteins, a new component was necessary and modeled by MCR-ALS. This new component could be assigned to the intermolecular beta-sheet, bridging protein molecules. MCR-ALS, therefore, unveiled a potential spectroscopic marker of protein aggregation and allowed a semiquantitative evaluation of its content. Insight into other structural rearrangements was also obtained.