Interactions Under Crowding Milieu: Chemical-Induced Denaturation of Myoglobin is Determined by the Extent of Heme Dissociation on Interaction with Crowders

Generally, in vivo function and structural changes are studied by probing proteins in a dilute solution under in vitro conditions, which is believed to be mimicking proteins in intracellular milieu. Earlier, thermal-induced denaturation of myoglobin, in the milieu of crowder molecule showed destabilization of the metal protein. Destabilization of protein by thermal-induced denaturation involves a large extrapolation, so, the reliability is questionable. This led us to measure the effects of macromolecular crowding on its stability by chemical-induced denaturation of the protein using probes like circular dichroism and absorption spectroscopy in the presence of dextran 70 and ficoll 70 at various pHs (acidic: 6.0, almost neutral: 7.0 and basic: 8.0). Observations showed that the degree of destabilization of myoglobin was greater due to ficoll 70 as compared to that of dextran 70 so it can be understood that the nature of the crowder or the shape of the crowder has an important role towards the stability of proteins. Additionally, the degree of destabilization was observed as pH dependent, however the pH dependence is different for different crowders. Furthermore, isothermal titration calorimetry and molecular docking studies confirmed that both the crowders (ficoll and dextran) bind to heme moiety of myoglobin and a single binding site was observed for each.

Photodynamic Opening of the Blood–Brain Barrier Using Different Photosensitizers in Mice

In a series of previous studies, we demonstrated that the photodynamic therapy (PDT), as a widely used tool for treatment of glioblastoma multiforme (GBM), also site-specifically opens the blood–brain barrier (BBB) in PDT-dose and age-related manner via reversible disorganization of the tight junction machinery. To develop the effective protocol of PDT-opening of the BBB, here we answer the question of what kind of photosensitizer (PS) is the most effective for the BBB opening. We studied the PDT-opening of the BBB in healthy mice using commercial photosensitizers (PSs) such as 5-aminolevulenic acid (5-ALA), aluminum phthalocyanine disulfonate (AlPcS), zinc phthalocyanine (ZnPc) and new synthetized PSs such as galactose functionalized ZnPc (GalZnPc). The spectrofluorimetric assay of Evans Blue albumin complex (EBAC) leakage and 3-D confocal imaging of FITC-dextran 70 kDa (FITCD) extravasation clearly shows a revisable and dose depended PDT-opening of the BBB to EBAC and FITCD associated with a decrease in presence of tight junction (TJ) in the vascular endothelium. The PDT effects on the BBB permeability, TJ expression and the fluorescent signal from the brain tissues are more pronounced in PDT-GalZnPc vs. PDT-5-ALA/AlPcS/ZnPc. These pre-clinical data are the first important informative platform for an optimization of the PDT protocol in the light of new knowledge about PDT-opening of the BBB for drug brain delivery and for the therapy of brain diseases.

Carbohydrate-Based Macromolecular Crowding-Induced Stabilization of Proteins: Towards Understanding the Significance of the Size of the Crowder

There are a large number of biomolecules that are accountable for the extremely crowded intracellular environment, which is totally different from the dilute solutions, i.e., the idealized conditions. Such crowded environment due to the presence of macromolecules of different sizes, shapes, and composition governs the level of crowding inside a cell. Thus, we investigated the effect of different sizes and shapes of crowders (ficoll 70, dextran 70, and dextran 40), which are polysaccharide in nature, on the thermodynamic stability, structure, and functional activity of two model proteins using UV-Vis spectroscopy and circular dichroism techniques. We observed that (a) the extent of stabilization of α-lactalbumin and lysozyme increases with the increasing concentration of the crowding agents due to the excluded volume effect and the small-sized and rod-shaped crowder, i.e., dextran 40 resulted in greater stabilization of both proteins than dextran 70 and ficoll 70; (b) structure of both the proteins remains unperturbed; and (c) enzymatic activity of lysozyme decreases with the increasing concentration of the crowder.