Evaluating the biomolecular interaction between delamanid/formulations and human serum albumin by fluorescence, CD spectroscopy and SPR: Effects on protein conformation, kinetic and thermodynamic parameters.

Delamanid is an anti-tuberculosis drug used for the treatment of drug-resistant tuberculosis. Since delamanid has a high protein bound potential, even patients with low albumin levels should experience high and rapid delamanid clearance. However, the interaction between delamanid and albumin should be better controlled to optimize drug efficacy. This study was designed to evaluate the binding characteristics of delamanid to human serum albumin (HSA) using various methods: fluorescence spectroscopy, circular dichroism (CD), surface plasmon resonance (SPR), and molecular docking simulation. The fluorescence emission band without any shift indicated the interaction was not affected by the polarity of the fluorophore microenvironment. The reduction of fluorescence intensity at 344 nm was proportional to the increment of delamanid concentration as a fluorescence quencher. UV-absorbance measurement at the maximum wavelength (λmax, 280 nm) was evaluated using inner filter effect correction. The HSA conformation change was explained by the intermolecular energy transfer between delamanid and HSA during complex formation. The study, which was conducted at temperatures of 298 K, 303 K, and 310 K, revealed a static quenching mechanism that correlated with a decreased of bimolecular quenching rate constant (kq) and binding constant (Ka) at increased temperatures. The Ka was 1.75-3.16 × 104 M-1 with a specific binding site with stoichiometry 1:1. The negative enthalpy change, negative entropy change, and negative Gibbs free energy change demonstrated an exothermic-spontaneous reaction while van der Waals forces and hydrogen bonds played a vital role in the binding. The molecular displacement approach and molecular docking confirmed that the binding occurred mainly in subdomain IIA, which is a hydrophobic pocket of HSA, with a theoretical binding free energy of -9.33 kcal/mol. SPR exhibited a real time negative sensorgram that resulted from deviation of the reflex angle due to ligand delamanid-HSA complex forming. The binding occurred spontaneously after delamanid was presented to the HSA surface. The SPR mathematical fitting model revealed that the association rate constant (kon) was 2.62 × 108 s-1M-1 and the dissociation rate constant (koff) was 5.65 × 10-3 s-1. The complexes were performed with an association constant (KA) of 4.64 × 1010 M-1 and the dissociation constant (KD) of 2.15 × 10-11 M. The binding constant indicated high binding affinity and high stability of the complex in an equilibrium. Modified CD spectra revealed that conformation of the HSA structure was altered by the presence of delamanid during preparation of the proliposomes that led to the reduction of secondary structure stabilization. This was indicated by the percentage decrease of α-helix. These findings are beneficial to understanding delamanid-HSA binding characteristics as well as the drug administration regimen.

In situ Phenotyping of Grapevine Root System Architecture by 2D or 3D Imaging: Advantages and Limits of Three Cultivation Methods.

The root system plays an essential role in the development and physiology of the plant, as well as in its response to various stresses. However, it is often insufficiently studied, mainly because it is difficult to visualize. For grapevine, a plant of major economic interest, there is a growing need to study the root system, in particular to assess its resistance to biotic and abiotic stresses, understand the decline that may affect it, and identify new ecofriendly production systems. In this context, we have evaluated and compared three distinct growing methods (hydroponics, plane, and cylindric rhizotrons) in order to describe relevant architectural root traits of grapevine cuttings (mode of grapevine propagation), and also two 2D- (hydroponics and rhizotron) and one 3D- (neutron tomography) imaging techniques for visualization and quantification of roots. We observed that hydroponics tubes are a system easy to implement but do not allow the direct quantification of root traits over time, conversely to 2D imaging in rhizotron. We demonstrated that neutron tomography is relevant to quantify the root volume. We have also produced a new automated analysis method of digital photographs, adapted for identifying adventitious roots as a feature of root architecture in rhizotrons. This method integrates image segmentation, skeletonization, detection of adventitious root skeleton, and adventitious root reconstruction. Although this study was targeted to grapevine, most of the results obtained could be extended to other plants propagated by cuttings. Image analysis methods could also be adapted to characterization of the root system from seedlings.

Effect of Ligands on HP-Induced Unfolding and Oligomerization of ß-Lactoglobulin.

To probe intermediate states during unfolding and oligomerization of proteins remains a major challenge. High pressure (HP) is a powerful tool for studying these problems, revealing subtle structural changes in proteins not accessible by other means of denaturation. Bovine ß-lactoglobulin (BLG), the main whey protein, has a strong propensity to bind various bioactive molecules such as retinol and resveratrol, two ligands with different affinity and binding sites. By combining in situ HP-small-angle neutron scattering (SANS) and HP-ultraviolet/visible absorption spectroscopy, we report the specific effects of these ligands on three-dimensional conformational and local changes in BLG induced by HP. Depending on BLG concentration, two different unfolding mechanisms are observed in situ under pressures up to ∼300 MPa: either a complete protein unfolding, from native dimers to Gaussian chains, or a partial unfolding with oligomerization in tetramers mediated by disulfide bridges. Retinol, which has a high affinity for the BLG hydrophobic cavity, significantly stabilizes BLG both in three-dimensional and local environments by shifting the onset of protein unfolding by ∼100 MPa. Increasing temperature from 30 to 37°C enhances the hydrophobic stabilization effects of retinol. In contrast, resveratrol, which has a low binding affinity for site(s) on the surface of the BLG, does not induce any significant effect on the structural changes of BLG due to pressure. HP treatment back and forth up to ∼300 MPa causes irreversible covalent oligomerization of BLG. Ab initio modeling of SANS shows that the oligomers formed from the BLG-retinol complex are smaller and more elongated compared to BLG without ligand or in the presence of resveratrol. By combining HP-SANS and HP-ultraviolet/visible absorption spectroscopy, our strategy highlights the crucial role of BLG hydrophobic cavity and opens up new possibilities for the structural determination of HP-induced protein folding intermediates and irreversible oligomerization.

Controlled Loading and Release of Beta-Lactoglobulin in Calcium-Polygalacturonate Hydrogels.

We show here how the structure of polygalacturonate (polyGalA) hydrogels cross-linked by Ca2+ cations via external gelation controls the loading and release rate of beta-lactoglobulin (BLG), a globular protein. Hydrogels prepared from a polyGalA/BLG solution are found to be similar to those obtained from a polyGalA solution in our previous study (Maire du Poset et al. Biomacromolecules 2019, 20 (7), 2864-2872): they exhibit similar transparencies and gradients of mechanical properties and polyGalA concentrations. The nominal BLG/polyGalA ratio of the mixtures is almost recovered within the whole mixed hydrogel despite such strong concentration gradients, except in the part of the hydrogels with the largest mesh size, where more BLG proteins are present. This gradient enables one to tune the amount of protein loaded within the hydrogel. At a local scale, the proteins are distributed evenly within the hydrogel network, as shown by small-angle neutron scattering (SANS). The release of proteins from hydrogels is driven by Fickian diffusion, and the release rate increases with the mesh size of the network, with a characteristic time of a few hours. The specific structure of these polysaccharide-based hydrogels allows for control of both the dosage and the release rate of the loaded protein and makes them good candidates for use as oral controlled-delivery systems.

A high pressure cell using metallic windows to investigate the structure of molecular solutions up to 600 MPa by small-angle neutron scattering.

We report on a high pressure (HP) cell designed for the determination of the structure of molecular solutions by small-angle neutron scattering (SANS). The HP cell is fitted up with two thick metallic windows that make the device very resistant under hydrostatic pressures up to 600 MPa (or 6 kbar). The metallic windows are removable, offering the possibility to adapt the HP cell to a given study with the pressure desired on an appropriate spatial range to study the structure of various molecular solutions by SANS. In this context, we report the absorption, transmission, and scattering properties of different metallic windows. Finally, we describe, as a proof of principle, the solution structure changes of myoglobin, a small globular protein.

Structural behaviour differences in low methoxy pectin solutions in the presence of divalent cations (Ca(2+) and Zn(2+)): a process driven by the binding mechanism of the cation with the galacturonate unit.

In this paper, we compare the interactions between low methoxy pectin (LMP) and either Ca(2+) or Zn(2+) in semi-dilute solutions. Intrinsic viscosity and turbidity measurements reveal that pectin-calcium solutions are more viscous, but yet less turbid, than pectin-zinc ones. To get a molecular understanding of the origin of this rather unexpected behavior, we further performed isothermal titration calorimetry, small angle neutron scattering experiments, as well as molecular dynamics simulations. Our results suggest that calcium cations induce the formation of a more homogeneous network of pectin than zinc cations do. The molecular dynamics simulations indicate that this difference could originate from the way the two cations bind to the galacturonate unit (Gal), the main component of LMP: zinc interacts with both carboxylate and hydroxyl groups of Gal, in a similar way to that described in the so-called egg-box model, whereas calcium only interacts with carboxylate groups. This different binding behavior seems to arise from the stronger interaction of water molecules with zinc than with calcium. Accordingly, galacturonate chains are more loosely associated with each other in the presence of Ca(2+) than with Zn(2+). This may improve their ability to form a gel, not only by dimerization, but also by the formation of point-like cross-links. Overall, our results show that zinc binds less easily to pectin than calcium does.

Myoglobin on silica: a case study of the impact of adsorption on protein structure and dynamics.

If protein structure and function changes upon adsorption are well documented, modification of adsorbed protein dynamics remains a blind spot, despite its importance in biological processes. The adsorption of metmyoglobin on a silica surface was studied by isotherm measurements, microcalorimetry, circular dichroïsm, and UV-visible spectroscopy to determine the thermodynamic parameters of protein adsorption and consequent structure modifications. The mean square displacement and the vibrational densities of states of the adsorbed protein were measured by elastic and inelastic neutron scattering experiments. A decrease of protein flexibility and depletion in low frequency modes of myoglobin after adsorption on silica was observed. Our results suggest that the structure loss itself is not the entropic driving force of adsorption.

The impact of high hydrostatic pressure on structure and dynamics of ß-lactoglobulin.

METHODS: Combining small-angle X-ray and neutron scattering measurements with inelastic neutron scattering experiments, we investigated the impact of high hydrostatic pressure on the structure and dynamics of ß-lactoglobulin (ßLG) in aqueous solution. BACKGROUND: ßLG is a relatively small protein, which is predominantly dimeric in physiological conditions, but dissociates to monomer below about pH3. RESULTS: High-pressure structural results show that the dimer-monomer equilibrium, as well as the protein-protein interactions, are only slightly perturbed by pressure, and ßLG unfolding is observed above a threshold value of 3000bar. In the same range of pressure, dynamical results put in evidence a slowing down of the protein dynamics in the picosecond timescale and a loss of rigidity of the ßLG structure. This dynamical behavior can be related to the onset of unfolding processes, probably promoted from water penetration in the hydrophobic cavity. GENERAL SIGNIFICANCE: Results suggest that density and compressibility of water molecules in contact with the protein are key parameters to regulate the protein flexibility.

Probing protein-membrane interactions using solid supported membranes.

Tethered bilayer lipid membranes have been used as a model system to mimic the interactions between the whey protein ß-lactoglobulin and a lipid interface. The approach allowed for a detailed study of the lipid-protein interactions, the results being of possible importance in food and cosmetic applications. For such applications, lipid-protein interactions and the interfacial behavior are vital factors in controlling and manipulating process conditions such as emulsion stabilization and gelification. Lipid composition as well as the structural properties of the protein governed their interactions, which were probed by a combination of surface plasmon spectroscopy, neutron reflectivity, and electrochemical impedance spectroscopy. Comparison of results obtained using native and a partially unfolded protein indicated that the protein preferentially forms loosely packed layers at the lipid interface.

Protein-lipid interactions at the air-water interface.

Protein-lipid interactions play an important role in a variety of fields, for example in pharmaceutical research, biosensing, or food science. However, the underlying fundamental processes that govern the interplay of lipids and proteins are often very complex and are therefore studied using model systems. Here, Langmuir monolayers were used to probe the interaction of a model protein with lipid films at the air-water interface. The protein beta-lactoglobulin (beta lg) is the major component in bovine milk serum, where it coexists with the milk fat globular membrane. During homogenization of milk, beta lg adsorbs to the interface of lipid fat globules and stabilizes the oil-in-water emulsion. pH and ionic strength of the subphase had a significant effect on the surface activity of the protein. Additionally, by using lipids with different charges, it could be shown that the interactions between beta lg and a phospholipid layer were driven by hydrophobic as well as by electrostatic interactions. beta lg preferentially interacted with phospholipids in an unfolded state. This could be either achieved by denaturation at the air-water interface or due to electrostatic interactions that weaken the intramolecular forces of the protein.

Structure of calcium and zinc pectinate films investigated by FTIR spectroscopy.

Calcium and zinc pectinate gels were prepared using a method which allowed calcium or zinc to diffuse from the cross-linking solution through a dialysis membrane to form a gel with amidated low-methoxyl pectin. The gel thus obtained was then dried, and the film structure was studied using FTIR spectroscopy as a function of the cation content (0%, 5%, 10%, and 15% w/v). Important consideration was given to the three functional groups (amide, carboxyl ester, and carboxylate groups) present in the pectin. When the zinc content was increased, the three wavenumber values corresponding to these three functional groups did not change significantly, while for calcium pectinate, the three wavenumber values were shifted profoundly when the amount of calcium ions changed. These results confirm that calcium ions could form stable interactions with carboxylate groups as described by the eggbox model [Grant, G.T.; Morris, E.R.; Rees, D.A.; Smith, P.J.C.; Tho, D. FEBS Lett.1973, 32, 195-198] while the lower coordination number of zinc does not permit a structured gel to develop.

High-pressure effects on horse heart metmyoglobin studied by small-angle neutron scattering.

Small-angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN3) at pressures up to 300 MPa. Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performed using various concentrations of MbN3, show that the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa. The value of the second virial coefficient of the protein solution indicates that the interactions between the molecules are always strongly repulsive even if their magnitude decreases with increasing pressure. Taking advantage of the pressure-induced contrast variation, these experiments allow the partial specific volume of MbN3 to be determined as a function of pressure. Its value decreases by 5.4% between atmospheric pressure and 300 MPa. In this pressure range the isothermal compressibility of hydrated MbN3 is found to be almost constant. Its value is (1.6 +/- 0.1) 10-4 MPa-1.