André Matagne

Differential scanning fluorimetry (DSF) is widely used to assess protein thermal stability and to compare protein variants, ligands, or formulations with limited sample consumption. Yet, the reproducibility of DSF-derived parameters across laboratories, instruments and analysis workflows remains poorly defined. Here, we report a two-phase interlaboratory benchmark using a shared V _H H sample across 26 laboratories, 28 instruments and seven DSF instrument models. In Phase 1, participants applied their routine acquisition and analysis workflows, whereas in Phase 2, acquisition conditions were harmonised. Datasets were analysed locally and then centrally reprocessed with MoltenProt. Participant-reported apparent melting temperatures ( T _m ) showed good within-instrument repeatability but moderate inter-instrument dispersion. Harmonisation of acquisition conditions did not substantially reduce this dispersion. Conversely, centralised reprocessing narrowed the distribution among retained datasets, indicating contributions from analysis workflows and data-acceptance criteria in addition to instrument-dependent factors. Comparison with differential scanning calorimetry (DSC) further showed that apparent T _m depended strongly on the fluorescence readout. For this V _H H, single-wavelength fluorescence at 350 nm showed the closest agreement with the DSC transition temperature, whereas the 350/330 nm ratio and barycentric mean generally yielded higher values. Furthermore, DSF-derived apparent unfolding enthalpies were more variable than apparent T _m . Together, these findings show that DSF can be highly repeatable within a defined instrument and workflow, but that quantitative comparability across instruments depends on instrument-specific effects, the choice of fluorescence readout and analysis strategy. Improved comparability will require transparent reporting of acquisition and analysis conditions, explicit data-quality criteria, and well-characterised reference materials to monitor instrument-dependent offsets.

When enzyme inhibition by either the product or excess substrate occurs, it is possible to determine the characteristic kinetic parameters based on [P]/t measurements, even when a large proportion of the substrate is converted. The advantages of various approaches are discussed. Most of them allow a good estimation of the V and K_m values. Conversely, the determination of K_p (product inhibition) and K_i (inhibition by excess substrate) can be more challenging. In the first case, determination of the type of inhibition requires more complex experiments that are beyond the scope of the present contribution. In the second, the inhibition constant K_i can only be roughly estimated. In an experimental approach, we compared the results obtained either with initial rate measurements or with 50 to 60% conversion of the substrate. Similar values of V and K_m were obtained. Measurements involving the conversion of a large proportion of substrate are particularly advantageous when the assay method is difficult or time-consuming, or when obtaining the substrate presents experimental difficulties or involves substantial costs.

In the chapters dealing with enzyme reactions, the authors of all Biochemistry textbooks and of even more specialized texts consider that the characteristic parameters (k_cat and K_m) must be determined under initial or steady-state rate conditions. This implies the transformation of a very limited proportion of substrate (at most 10–20%) or a continuous recording of the product or substrate concentration vs. time. Both options can present practical difficulties. Is it possible to get around these very stringent conditions? Here we show that in the most favourable cases up to 70% of the substrate can be converted resulting in systematic errors on the parameters (that can easily be taken account of) if the simple Henri-Michaelis–Menten equation is utilised. Alternatively, the integrated form of the same equation directly yields excellent estimates of the same parameters. Our observations should greatly facilitate the task of researchers who study systems in which measurements of the reaction progress are painstaking or when substrate concentrations close to the detection limit must be used. The general conclusion is that it is not always absolutely necessary to determine initial or steady-state rates to obtain reliable estimations of the enzyme kinetic parameters..

Human peroxiredoxin-5 (PRDX5) is a unique redox-sensitive protein that plays a dual role in brain ischemia-reperfusion injury. While intracellular PRDX5 has been reported to act as a neuroprotective antioxidative enzyme by scavenging peroxides, once released extracellularly from necrotic brain cells, the protein aggravates neural cell death by inducing expression of proinflammatory cytokines in macrophages through activation of Toll-like receptor (TLR) 2 (TLR2) and 4 (TLR4). Although recent evidence showed that PRDX5 was able to interact directly with TLR4, little is known regarding the role of the cysteine redox state of PRDX5 on its DAMP function. To gain insights into the role of PRDX5 redox-active cysteine residues in the TLR4-dependent proinflammatory activity of the protein, we used a recombinant human PRDX5 in the disulfide (oxidized) form and a mutant version lacking the peroxidatic cysteine, as well as chemically reduced and hyperoxidized PRDX5 proteins. We first analyzed the oxidation state and oligomerization profile by Western blot, mass spectrometry, and SEC-MALS. Using ELISA, we demonstrate that the disulfide bridge between the enzymatic cysteines is required to allow improved TLR4-dependent IL-8 secretion. Moreover, single-molecule force spectroscopy experiments revealed that TLR4 alone is not sufficient to discriminate the different PRDX5 redox forms. Finally, flow cytometry binding assays show that disulfide PRDX5 has a higher propensity to bind to the surface of living TLR4-expressing cells than the mutant protein. Taken together, these results demonstrate the importance of the redox state of PRDX5 cysteine residues on TLR4-induced inflammation.

The complex topologies of large multi-domain globular proteins make the study of their folding and assembly particularly demanding. It is often characterized by complex kinetics and undesired side reactions, such as aggregation. The structural simplicity of tandem-repeat proteins, which are characterized by the repetition of a basic structural motif and are stabilized exclusively by sequentially localized contacts, has provided opportunities for dissecting their folding landscapes. In this study, we focus on the Erwinia chrysanthemi pectin methylesterase (342 residues), an all-β pectinolytic enzyme with a right-handed parallel β-helix structure. Chemicals and pressure were chosen as denaturants and a variety of optical techniques were used in conjunction with stopped-flow equipment to investigate the folding mechanism of the enzyme at 25 °C. Under equilibrium conditions, both chemical- and pressure-induced unfolding show two-state transitions, with average conformational stability (ΔG° = 35 ± 5 kJ·mol⁻¹) but exceptionally high resistance to pressure (P_m = 800 ± 7 MPa). Stopped-flow kinetic experiments revealed a very rapid (τ < 1 ms) hydrophobic collapse accompanied by the formation of an extended secondary structure but did not reveal stable tertiary contacts. This is followed by three distinct cooperative phases and the significant population of two intermediate species. The kinetics followed by intrinsic fluorescence shows a lag phase, strongly indicating that these intermediates are productive species on a sequential folding pathway, for which we propose a plausible model. These combined data demonstrate that even a large repeat protein can fold in a highly cooperative manner.

Among various factors, the direct environment (e.g. pH, buffer components, salts, additives, etc.…) is known to have a crucial effect on both the stability and activity of proteins. In particular, proper buffer and pH conditions can improve their stability and function significantly during purification, storage and handling, which is highly relevant for both academic and industrial applications. It can also promote data reproducibility, support the interpretation of experimental results and, finally, contribute to our general understanding of the biophysical properties of proteins. In this study, we have developed a high throughput screen of 158 different buffers/pH conditions in which we evaluated: (i) the protein stability, using differential scanning fluorimetry and (ii) the protein function, using either enzymatic assays or binding activity measurements, both in an automated manner. The modular setup of the screen allows for easy implementation of other characterization methods and parameters, as well as additional test conditions. The buffer/pH screen was validated with five different proteins used as models, i.e. two active-site serine β-lactamases, two metallo-β-lactamases (one of which is only active as a tetramer) and a single-domain dromedary antibody fragment (V_HH or nanobody). The formulation screen allowed automated and fast determination of optimum buffer and pH profiles for the tested proteins. Besides the determination of the optimum buffer and pH, the collection of pH profiles of many different proteins may also allow to delineate general concepts to understand and predict the relationship between pH and protein properties.

KPC enzymes are the most common class A carbapenemases globally diffused. The peculiarity of this family of β-lactamases is represented by their ability to hydrolyse all classes of β-lactams, including carbapenems, posing a serious problem to public health. In the present study, seven laboratory mutants of KPC-3 (D228S, D228W, D228M, D228K, D228L, D228I and D228G) were generated by site-saturation mutagenesis to explore the role of residue 228, a non-active site residue. Compared to KPC-3, the seven mutants showed evident differences in k_cat and K_m values calculated for some penicillins, cephalosporins and carbapenems. In particular, D228S and D228M showed a significant increase of K_m values for cefotaxime and ceftazidime. Circular dichroism (CD) experiments have demonstrated that substitution at position 228 does not affect the secondary structure of the mutants. Molecular dynamics (MD) simulations were performed on KPC-3, D228S and D228M uncomplexed and complexed with cefotaxime (substrate). Although the residue 228 is located far from the active site, between α11 helix and β7 sheet in the opposite site of the Ω-loop, amino acid substitution at this position generates mechanical effects in the active site resulting in enzyme activity changes.

As it forms water‐filled channel in the mitochondria outer membrane and diffuses essential metabolites such as NADH and ATP, the voltage‐dependent anion channel (VDAC) protein family plays a central role in all eukaryotic cells. In comparison with their mammalian homologues, little is known about the structural and functional properties of plant VDACs. In the present contribution, one of the two VDACs isoforms of Solanum tuberosum, stVDAC36, has been successfully overexpressed and refolded by an in‐house method, as demonstrated by the information on its secondary and tertiary structure gathered from circular dichroism and intrinsic fluorescence. Cross‐linking and molecular modeling studies have evidenced the presence of dimers and tetramers, and they suggest the formation of an intermolecular disulfide bond between two stVDAC36 monomers. The pore‐forming activity was also assessed by liposome swelling assays, indicating a typical pore diameter between 2.0 and 2.7 nm. Finally, insights about the ATP binding inside the pore are given by docking studies and electrostatic calculations.