Caging and Photoactivation in Single-Molecule Forster Resonance Energy Transfer Experiments

Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Forster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor acceptor pairs during solution-based smFRET. We use this "caged FRET" methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules

Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc.

In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a Kd of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc)

Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIA Glc

In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)–LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a K(d) of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc)

Traditional Cultivars Influence on Physical and Engineering Properties of Rice from the Cauvery Deltaic Region of Tamil Nadu

Standard unit operations/equipment have not evolved for the traditional rice varieties of the Cauvery Deltaic region of Tamil Nadu. The fame of traditional rice is increasing nowadays owing to its health benefits. Non-standard unit operations may cause rice grains to crack during milling, accumulating more broken rice and yields in products of inferior quality. As a result, research into the physical properties of rice is crucial for the development of rice processing equipment that minimizes post-harvest losses during milling. Hence, an assessment was made to evaluate 30 traditional rice cultivars on their Physical (grain length, width, thickness, shape, and size), gravimetric (bulk, true, tapped density, porosity, Carr’s index, and Hausner ratio), and engineering characteristics (equivalent, arithmetic, square mean, and geometric mean diameter) using standard protocols, with the goal of reviving and preserving older varieties. The results from the analysis showed significant variations (p 2, respectively. Of the 30 varieties, 28 were under the high amylose category, and 2 belonged to the intermediate type. The Pearson correlation was established to study the interrelationships between the dimensions and engineering properties. Principal component analysis (PCA) reduced the dimensionality of 540 data into five principal components (PC), which explained 95.7% of the total variance. These findings suggest that it is possible to revive old landraces through careful selection and analysis of these properties. The superior characteristics of these traditional varieties can be further evaluated for breeding programs in order to improve the cultivation of these cherished rice landraces to enhance nutritional security

Unravelling the metabolomic diversity of pigmented and non-pigmented traditional rice from Tamil Nadu, India

Abstract Rice metabolomics is widely used for biomarker research in the fields of pharmacology. As a consequence, characterization of the variations of the pigmented and non-pigmented traditional rice varieties of Tamil Nadu is crucial. These varieties possess fatty acids, sugars, terpenoids, plant sterols, phenols, carotenoids and other compounds that plays a major role in achieving sustainable development goal 2 (SDG 2). Gas-chromatography coupled with mass spectrometry was used to profile complete untargeted metabolomics of Kullkar (red colour) and Milagu Samba (white colour) for the first time and a total of 168 metabolites were identified. The metabolite profiles were subjected to data mining processes, including principal component analysis (PCA), Orthogonal Partial Least Square Discrimination Analysis (OPLS-DA) and Heat map analysis. OPLS-DA identified 144 differential metabolites between the 2 rice groups, variable importance in projection (VIP) ≥ 1 and fold change (FC) ≥ 2 or FC ≤ 0.5. Volcano plot (64 down regulated, 80 up regulated) was used to illustrate the differential metabolites. OPLS-DA predictive model showed good fit (R2X = 0.687) and predictability (Q2 = 0.977). The pathway enrichment analysis revealed the presence of three distinct pathways that were enriched. These findings serve as a foundation for further investigation into the function and nutritional significance of both pigmented and non-pigmented rice grains thereby can achieve the SDG 2

Data_Sheet_1_Metabolomic analysis for disclosing nutritional and therapeutic prospective of traditional rice cultivars of Cauvery deltaic region, India.pdf

Traditional rice is gaining popularity worldwide due to its high nutritional and pharmaceutical value, as well as its high resistance to abiotic and biotic stresses. This has attracted significant attention from breeders, nutritionists, and plant protection scientists in recent years. Hence, it is critical to investigate the grain metabolome to reveal germination and nutritional importance. This research aimed to explore non-targeted metabolites of five traditional rice varieties, viz., Chinnar, Chithiraikar, Karunguruvai, Kichili samba, and Thooyamalli, for their nutritional and therapeutic properties. Approximately 149 metabolites were identified using the National Institute of Standards and Technology (NIST) library and Human Metabolome Database (HMDB) and were grouped into 34 chemical classes. Major classes include fatty acids (31.1–56.3%), steroids and their derivatives (1.80–22.4%), dihydrofurans (8.98–11.6%), prenol lipids (0.66–4.44%), organooxygen compounds (0.12–6.45%), benzene and substituted derivatives (0.53–3.73%), glycerolipids (0.36–2.28%), and hydroxy acids and derivatives (0.03–2.70%). Significant variations in metabolite composition among the rice varieties were also observed through the combination of univariate and multivariate statistical analyses. Principal component analysis (PCA) reduced the dimensionality of 149 metabolites into five principle components (PCs), which explained 96% of the total variance. Two clusters were revealed by hierarchical cluster analysis, indicating the distinctiveness of the traditional varieties. Additionally, a partial least squares-discriminant analysis (PLS-DA) found 17 variables important in the projection (VIP) scores of metabolites. The findings of this study reveal the biochemical intricate and distinctive metabolomes of the traditional therapeutic rice varieties. This will serve as the foundation for future research on developing new rice varieties with traditional rice grain metabolisms to increase grain quality and production with various nutritional and therapeutic benefits.</p

YidC assists the stepwise and stochastic folding of membrane proteins

How chaperones, insertases and translocases facilitate insertion and folding of complex cytoplasmic proteins into cellular membranes is not fully understood. Here, we utilize single-molecule force spectroscopy to observe YidC, a transmembrane chaperone/insertase, sculpting the folding trajectory of the polytopic α-helical membrane protein lactose permease (LacY). In the absence of YidC, unfolded LacY inserts individual structural segments into the membrane; however, misfolding dominates the process so that folding cannot be completed. YidC prevents LacY from misfolding by stabilizing the unfolded state from which LacY inserts structural segments stepwise into the membrane until folding is completed. During stepwise insertion, YidC and membrane together stabilize the transient folds. Remarkably, the order of insertion of structural segments is stochastic, thereby indicating that LacY can fold along variable pathways towards the native structure. Since YidC is essential in membrane protein biogenesis and LacY a paradigm for the major facilitator superfamily, our observations have general relevance