Study of changes in folding/unfolding properties and stability of Arabidopsis thaliana MYB12 transcription factor following UV-B exposure in vitro.

Flavonoids mostly protect plant cells from the harmful effects of UV-B radiation from the sun. In plants, the R2R3-subfamily of the MYB transcription factor, MYB12, is a key inducer of the biosynthesis of flavonoids. Our study involves the biophysical characterization of Arabidopsis thaliana MYB12 protein (AtMYB12) under UV-B exposure in vitro. Tryptophan fluorescence studies using recombinant full-length AtMYB12 (native) and the N-terminal truncated versions (first N-terminal MYB domain absent in AtMYB12Δ1, and both the first and second N-terminal MYB domains absent in AtMYB12Δ2) have revealed prominent alteration in the tryptophan microenvironment in AtMYB12Δ1 and AtMYB12Δ2 protein as a result of UV-B exposure as compared with the native AtMYB12. Bis-ANS binding assay and urea-mediated denaturation profiling showed an appreciable change in the structural conformation in AtMYB12Δ1 and AtMYB12Δ2 proteins as compared with the native AtMYB12 protein following UV-B irradiation. UV-B-treated AtMYB12Δ2 showed a higher predisposition of aggregate formation in vitro. CD spectral analyses revealed a decrease in α-helix percentage with a concomitant increase in random coiled structure formation in AtMYB12Δ1 and AtMYB12Δ2 as compared to native AtMYB12 following UV-B treatment. Overall, these findings highlight the critical function of the N-terminal MYB domains in maintaining the stability and structural conformation of the AtMYB12 protein under UV-B stress in vitro.

Structural Water Stabilizes Protein Motifs in Liquid Protein Phase: The Folding Mechanism of Short ß-Sheets Coupled to Phase Transition.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.

In Situ Monitored Vortex Fluidic-Mediated Protein Refolding/Unfolding Using an Aggregation-Induced Emission Bioprobe.

Protein folding is important for protein homeostasis/proteostasis in the human body. We have established the ability to manipulate protein unfolding/refolding for ß-lactoglobulin using the induced mechanical energy in the thin film microfluidic vortex fluidic device (VFD) with monitoring as such using an aggregation-induced emission luminogen (AIEgen), TPE-MI. When denaturant (guanidine hydrochloride) is present with ß-lactoglobulin, the VFD accelerates the denaturation reaction in a controlled way. Conversely, rapid renaturation of the unfolded protein occurs in the VFD in the absence of the denaturant. The novel TPE-MI reacts with exposed cysteine thiol when the protein unfolds, as established with an increase in fluorescence intensity. TPE-MI provides an easy and accurate way to monitor the protein folding, with comparable results established using conventional circular dichroism. The controlled VFD-mediated protein folding coupled with in situ bioprobe AIEgen monitoring is a viable methodology for studying the denaturing of proteins.

Effects of Protein Unfolding on Aggregation and Gelation in Lysozyme Solutions.

In this work, we investigate the role of folding/unfolding equilibrium in protein aggregation and formation of a gel network. Near the neutral pH and at a low buffer ionic strength, the formation of the gel network around unfolding conditions prevents investigations of protein aggregation. In this study, by deploying the fact that in lysozyme solutions the time of folding/unfolding is much shorter than the characteristic time of gelation, we have prevented gelation by rapidly heating the solution up to the unfolding temperature (~80 °C) for a short time (~30 min.) followed by fast cooling to the room temperature. Dynamic light scattering measurements show that if the gelation is prevented, nanosized irreversible aggregates (about 10-15 nm radius) form over a time scale of 10 days. These small aggregates persist and aggregate further into larger aggregates over several weeks. If gelation is not prevented, the nanosized aggregates become the building blocks for the gel network and define its mesh length scale. These results support our previously published conclusion on the nature of mesoscopic aggregates commonly observed in solutions of lysozyme, namely that aggregates do not form from lysozyme monomers in their native folded state. Only with the emergence of a small fraction of unfolded proteins molecules will the aggregates start to appear and grow.

Role of His101 in the Protein Folding/Unfolding of a Goose-Type Lysozyme from Ostrich (Struthio camelus) Egg White.

To understand the role of His101 in protein structure stabilization of goose-type (G-type) lysozyme, we conducted thermal unfolding/refolding experiments using native G-type lysozyme from ostrich egg white (nOEL), the recombinant G-type lysozyme (rOEL), and the mutant lysozyme, in which His101 is mutated to alanine (H101A-OEL). Thermal stability on lytic activity and in-gel refolding experiments provided similar profiles for all three OELs. Circular dichroism (CD) spectroscopy was used to determine the secondary structure of three OELs as a function of temperature. Unfolding/refolding experiments (30-90 °C) monitored by CD spectroscopy revealed an unfolding transition at 65-67 °C and a complete refolding at almost the same temperature. Notably, a slightly lower thermal stability was observed for H101A-OEL, corresponding to the calculated difference in transition free energy of thermal unfolding (∆∆G m) between rOEL and H101A-OEL of -0.63 kcal/mol. To assess the effects of H101A mutation on the electrostatic behavior, we examined the pH-activity profile of the three OELs. nOEL and rOEL exhibit bimodal relationship between pH and lytic activity showing optima at pH 3.0 and 7.0, while optima for H101A-OEL activity were pH 4.0 and 6.0. Electrostatic environment surrounding His101 was affected by the H101A mutation resulting in the slightly lower thermal stability.