Osmolyte induced protein stabilization: modulation of associated water dynamics might be a key factor.

The mechanism of protein stabilization by osmolytes remains one of the most important and long-standing puzzles. The traditional explanation of osmolyte-induced stability through the preferential exclusion of osmolytes from the protein surface has been seriously challenged by the observations like the concentration-dependent reversal of osmolyte-induced stabilization/destabilization. The more modern explanation of protein stabilization/destabilization by osmolytes considers an indirect effect due to osmolyte-induced distortion of the water structure. It provides a general mechanism, but there are numerous examples of protein-specific effects, i.e., a particular osmolyte might stabilize one protein, but destabilize the other, that could not be rationalized through such an explanation. Herein, we hypothesized that osmolyte-induced modulation of associated water might be a critical factor in controlling protein stability in such a medium. Taking different osmolytes and papain as a protein, we proved that our proposal could explain protein stability in osmolyte media. Stabilizing osmolytes rigidify associated water structures around the protein, whereas destabilizing osmolytes make them flexible. The strong correlation between the stability and the associated water dynamics, and the fact that such dynamics are very much protein specific, established the importance of considering the modulation of associated water structures in explaining the osmolyte-induced stabilization/destabilization of proteins. More interestingly, we took another protein, bromelain, for which a traditionally stabilizing osmolyte, sucrose, acts as a stabilizer at higher concentrations but as a destabilizer at lower concentrations. Our proposal successfully explains such observations, which is probably impossible by any known mechanisms. We believe this report will trigger much research in this area.

Understanding the intricacy of protein in hydrated deep eutectic solvent: Solvation dynamics, conformational fluctuation dynamics, and stability.

Deep eutectic solvents (DESs) are potential biocatalytic media due to their easy preparation, fine-tuneability, biocompatibility, and most importantly, due to their ability to keep protein stable and active. However, there are many unanswered questions and gaps in our knowledge about how proteins behave in these alternate media. Herein, we investigated solvation dynamics, conformational fluctuation dynamics, and stability of human serum albumin (HSA) in 0.5 Acetamide/0.3 Urea/0.2 Sorbitol (0.5Ac/0.3Ur/0.2Sor) DES of varying concentrations to understand the intricacy of protein behaviour in DES. Our result revealed a gradual decrease in the side-chain flexibility and thermal stability of HSA beyond 30 % DES. On the other hand, the associated water dynamics around domain-I of HSA decelerate only marginally with increasing DES content, although viscosity rises considerably. We propose that even though macroscopic solvent properties are altered, a protein feels only an aqueous type of environment in the presence of DES. This is probably the first experimental study to delineate the role of the associated water structure of the enzyme for maintaining its stability inside DES. Although considerable effort is necessary to generalize such claims, it might serve as the basis for understanding why proteins remain stable and active in DES.

Does Microsecond Active-Site Dynamics Primarily control Proteolytic Activity of Bromelain? Clues from Single Molecular Level Study with a Denaturant, a Stabilizer and a Macromolecular Crowder.

Proteins are dynamic entity with various molecular motions at different timescale and length scale. Molecular motions are crucial for the optimal function of an enzyme. It seems intuitive that these motions are crucial for optimal enzyme activity. However, it is not easy to directly correlate an enzyme's dynamics and activity due to biosystems' enormous complexity. amongst many factors, structure and dynamics are two prime aspects that combinedly control the activity. Therefore, having a direct correlation between protein dynamics and activity is not straightforward. Herein, we observed and correlated the structural, functional, and dynamical responses of an industrially crucial proteolytic enzyme, bromelain with three versatile classes of chemicals: GnHCl (protein denaturant), sucrose (protein stabilizer), and Ficoll-70 (macromolecular crowder). The only free cysteine (Cys-25 at the active-site) of bromelain has been tagged with a cysteine-specific dye to unveil the structural and dynamical changes through various spectroscopic studies both at bulk and at the single molecular level. Proteolytic activity is carried out using casein as the substrate. GnHCl and sucrose shows remarkable structure-dynamics-activity relationships. Interestingly, with Ficoll-70, structure and activity are not correlated. However, microsecond dynamics and activity are beautifully correlated in this case also. Overall, our result demonstrates that bromelain dynamics in the microsecond timescale around the active-site is probably a key factor in controlling its proteolytic activity.

Synthesis, Characterization, Photocatalytic and Sensing Properties of Mn-Doped ZnO Nanoparticles.

In this paper, Mn-doped ZnO nanoparticles (0 to 10 mol% Mn) were synthesized by facile low-temperature aqueous solution process and characterized by several techniques such as field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), UV-visible and Raman-scattering spectroscopy. The SEM studies confirmed that the synthesized nanoparticles are grown in high density and increase in Mn content was found to have a significant effect on the morphologies of ZnO nanoparticles. The XPS studies established the structural variation of the samples with the change in dopant concentration and its oxidation state. XPS probe the existence of impurity phases in the as-synthesized samples. The results indicate further that hexagonal wurtzite structure of ZnO undergoes distortion with the increase in the dopant concentration. Also, with the increase in the dopant concentration, the blue-shift was observed in the UV-vis. spectra. Photocatalytic and chemicals sensing performances of these nanomaterials have been investigated by subjecting them to photocatalytic degradation of methyl orange (MO) under UV irradiation and for the detection of picric acid (PA) in aqueous solutions. Mn doped ZnO samples were found to be more efficient in catalyzing the MO degradation than pure ZnO. 5 mol% Mn doped ZnO nanomaterials were studied to use as fluorescence sensor for the detection of PA and the observed detection limit was found to be 2.5 µM.

Iron Oxide Nanoparticles as Potential Scaffold for Photocatalytic and Sensing Applications.

Herein, we report photocatalytic and fluorescence sensing applications of iron oxide (α-Fe2O3) nanoparticles synthesized by facile low-temperature simple solution process. The synthesized nanoparticles were characterized by several techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM) attached with energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM), UV-visible spectroscopy and fluorescence spectroscopy. The detailed analysis revealed that the synthesized nanoparticles were well-crystalline, grown in very high density and possessing rhombohedral α-Fe2O3 crystal structure. The prepared nanoparticles were used as efficient scaffold for photocatalytic and sensing applications. The detailed photocatalysis results revealed that in presence of an appropriate amount of α-Fe2O3 nanoparticles as photocatalyst, a significant dye degradation of methyl orange (MO) was observed in 140 min. In addition, the fabricated florescence sensor based on α-Fe2O3 nanoparticles exhibited a low detection limit of ∼3.33 µM/L towards picric acid. The observed results clearly confirmed that the synthesized α-Fe2O3 nanoparticles are potential scaffold for photocatalysis and sensing applications.

Purification and characterization of a lectin with high hemagglutination property isolated from Allium altaicum.

A lectin was purified from the leaves of Allium altaicum and corresponding gene was cloned. The lectin namely Allium altaicum agglutinin (AAA) was ~24 kDa homodimeric protein and similar to a typical garlic leaf lectin. It was synthesized as 177 amino acid residues pre-proprotein, which consisted of 28 and 43 amino acid long N and C-terminal signal peptides, respectively. The plant expressed this protein more in scapes and flowers in comparison to the bulbs and leaves. Hemagglutination activity (with rabbit erythrocytes) was 1,428 fold higher as compared to Allium sativum leaf agglutinin (ASAL) although, the insecticidal activity against cotton aphid (Aphis gossypii) was relatively low. Glycan array revealed that AAA had higher affinity towards GlcAb1-3Galb as compared to ASAL. Homology analysis showed 57-94% similarity with other Allium lectins. The mature protein was expressed in E. coli as a fusion with SUMO peptide in soluble and biologically active form. Recombinant protein retained high hemagglutination activity.