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The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors.

Infections caused by Staphylococcus aureus depend on its ability to acquire nutrients. One essential nutrient is iron, which is obtained from the heme of the human host hemoglobin (Hb) through a protein machinery called Iron-regulated surface determinant (Isd) system. IsdB is the protein in charge of heme extraction from Hb, which is the first step of the chain of events leading to iron transfer to the bacterium cell interior. In order to elucidate the molecular events leading from the formation of the initial IsdB:Hb complex to heme extraction, we use time-resolved X-ray solution scattering (TR-XSS) in combination with rapid mixing triggering. We succeed in defining the stoichiometry of IsdB:Hb binding and in describing the kinetics of the subsequent structural changes. The presented approach is potentially applicable to unveil the complex kinetic pathways generated by protein-protein interaction in different biological systems. Iron-regulated surface determinant B (IsdB) from Staphylococcus aureus extracts heme from human hemoglobin, which is the first iron transfer step to the bacterial cell. Here, IsdB functional dynamics are explored with X-ray solution scattering techniques including time-resolved WAXS.

Allantoin, the natural end product of purine catabolism in mammals, is non-enzymatically produced from the scavenging of reactive oxygen species through the degradation of uric acid. Levels of allantoin in biological fluids are sensitively influenced by the presence of free radicals, making this molecule a candidate marker of acute oxidative stress in clinical analyses. With this aim, we exploited allantoinase—the enzyme responsible for allantoin hydrolization in plants and lower organisms—for the development of a biosensor exploiting a fast enzymatic-chemical assay for allantoin quantification. Recombinant allantoinase was entrapped in a wet nanoporous silica gel matrix and its structural properties, function, and stability were characterized through fluorescence spectroscopy and circular dichroism measurements, and compared to the soluble enzyme. Physical immobilization in silica gel minimally influences the structure and the catalytic efficiency of entrapped allantoinase, which can be reused several times and stored for several months with good activity retention. These results, together with the relative ease of the sol-gel preparation and handling, make the encapsulated allantoinase a good candidate for the development of an allantoin biosensor.

Iron is an essential micronutrient for bacterial growth and plays a crucial role in various physiological processes, including motility. This study investigates the influence of iron availability on the behaviour of Staphylococcus aureus , a significant human pathogen, using a nanomotion sensing technique. By correlating nanoscale vibrations with metabolically related behaviour, this sensor offers real-time insight into the activity of biological specimens from single proteins to living bacteria and mammalian cells. S. aureus cultures were grown under iron-repleted and iron-depleted conditions, and their nano-scale motility was monitored in real time. We compared the activity of wild type S. aureus with an isogenic deletion mutant, in which siderophore production was abrogated. Our results demonstrate distinct motility patterns of the two strains in response to varying iron concentrations, including different outlines of their activity. We identified the fingerprint of the iron intake pathway for the two strains: in the presence of iron, while the wild-type strain followed an exponential growth pattern, the mutant suspended its growth. Remarkably, in presence of iron, both strains showed some coordinated oscillations at well-defined frequencies, which could be related to specific behaviour of S. aureus . These results were corroborated by the phenotypic assays performed through optical microscopy and conventional growth methods. Such findings highlight the potential of nanomotion sensing, a powerful tool to study cellular vibrations and to investigate the complex relationship between iron homeostasis and S. aureus activity, suggesting this metabolic route as a possible fruitful target for the development of novel antibacterials.

Protein-based biosensors require controlled and site-selective functionalization strategies to enable stable and oriented immobilization without compromising protein structure and signal transduction efficiency. We evaluated a chemoselective linchpin-directed modification (LDM) approach targeting Lys–His pairs as a tool for site-specific labeling of the model fluorescent biosensor green fluorescent protein (GFP). LDM molecules with variable spacer lengths were prepared, and a structure-guided computational workflow was implemented to map Lys–His distances on the protein and identify candidate modification sites. Experimental validation by UV-Vis spectroscopy and mass spectrometry demonstrated efficient conjugation and a final degree of labeling close to unity, consistent with single-site modification, with all LDM molecules selectively targeting the same histidine residue (His181), independently of spacer length. Structural analysis revealed that this residue is located within an accessible internal cavity that favors productive interactions with the reactive group. Importantly, the modification preserves GFP fluorescence and pH response, confirming retention of sensing functionality. These results demonstrate that LDM enables selective modification not only of surface residues, but also of structurally guided, non-surface residues. This approach provides the proof of concept of a new, promising strategy for the controlled functionalization and immobilization of protein-based biosensors.

In the pharmaceutical field, lipid-based nanoparticles are extensively used for drug or vaccine delivery, particularly for treating respiratory disorders. However, their physico-chemical instability, particularly associated with lipid degradation through hydrolysis or oxidation, can affect their encapsulation properties. To monitor the stability of lipid-based formulations over time, we prepared acrylodan-labeled bovine serum albumin (here called albuminodan), and showed it is a fluorescent biosensor capable of concomitantly detect phospholipids as well as their degradation products, i.e., fatty acids and lysophospholipids. We demonstrated that this tool can be used to follow the distribution of lipids in an aqueous phase and hence could be suitable to characterize the hydrolysis of phospholipids in a lipid-based formulation to monitor the stability of nanoparticles.

Cysteine biosynthesis is increasingly recognized as a critical determinant of bacterial virulence, highlighting this pathway as a promising reservoir of novel antimicrobial targets. In , however, the molecular basis of cysteine production has only recently begun to emerge. Here, we identify PA3816 as the major serine acetyltransferase (PaCysE), the enzyme responsible for generating the activated serine intermediate that feeds -acetylserine sulfhydrylase-mediated cysteine synthesis. Through a combination of biochemical and genetic approaches, we demonstrate that PaCysE efficiently catalyzes L-serine acetylation , and in turn, deletion mutants exhibit cysteine auxotrophy, underscoring its essential contribution to -acetylserine production. Notably, PaCysE is less sensitive to feedback inhibition by cysteine and does not appear to form the canonical cysteine synthase complex, suggesting a regulatory architecture that diverges from well-characterized orthologs. Loss of PaCysE function has broad physiological consequences, including enhanced biofilm formation, reduced pyocyanin production, and attenuated infectivity in an animal model, linking cysteine biosynthesis directly to pathogen fitness. Finally, we identify a thiazole derivative that inhibits PaCysE activity (IC ≈ 30 µM) and suppresses bacterial growth in a cysteine-dependent manner, providing a proof-of-concept for therapeutically targeting this pathway.

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent α₂β₂ complex catalyzing the last two steps of tryptophan biosynthesis in bacteria, plants and fungi. Structural, dynamic and functional studies, carried out over more than 40 years, have unveiled that: (1) α- and β-active sites are separated by about 20 Å and communicate via the selective stabilization of distinct conformational states, triggered by the chemical nature of individual catalytic intermediates and by allosteric ligands; (2) indole, formed at α-active site, is intramolecularly channeled to the β-active site; and (3) naturally occurring as well as genetically generated mutants have allowed to pinpoint functional and regulatory roles for several individual amino acids. These key features have made tryptophan synthase a text-book case for the understanding of the interplay between chemistry and conformational energy landscapes.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus . This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein–protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico / in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

Iron surface determinant B (IsdB), a Staphylococcus aureus (SA) surface protein involved in both heme iron acquisition from host hemoglobin (Hb) and bacterial adhesion, is a proven virulence factor that can be targeted for the design of antibacterial molecules or vaccines. Recent single-molecule experiments on IsdB interaction with cell adhesion factors revealed an increase of the complex lifetime upon applying a stronger force (catch bond); this was suggested to favor host invasion under shear stress. An increased bond strength under mechanical stress was also detected by Atomic Force Spectroscopy (AFS) for the interaction between IsdB and Hb. Structural information on the underlying molecular mechanisms at the basis of this behaviour in IsdB-based complexes is missing. Here, we show that the single point mutation of Pro173 in the IsdB domain responsible for Hb binding, which weakens the IsdB:Hb interaction without hampering heme extraction, totally abolishes the previously observed behavior. Remarkably, Pro173 does not directly interact with Hb, but undergoes cis–trans isomerization upon IsdB:Hb complex formation, coupled to folding-upon binding of the corresponding protein loop. Our results suggest that these events might represent the molecular basis for the stress-dependence of bond strength observed for wild type IsdB, shedding light on the mechanisms that govern the capability of SA to infect host cells.