Notes

And,N'-bicarbazole-benzothiadiazole-based conjugated porous organic and natural polymer bonded regarding sensitive air species age group in are living tissue.
Finally, we describe a straightforward experimental protocol on the determination of crowding sensitivity in a buffer and cell.The peptide/protein pair, SpyTag/SpyCatcher, which is derived from split immunoglobulin-like collagen adhesin domain (CnaB2) from Streptococcus pyogenes, can spontaneously form a stable Lys-Asp isopeptide bond under physiological conditions. This enabling technology- also known as genetically encoded click chemistry owing to its marked efficiency and specificity-has led to a variety of applications in protein engineering, materials science and synthetic biology in recent years. In this chapter, we discuss the use of SpyTag/SpyCatcher chemistry to create nonlinear protein architectures and materials, with emphasis on its role in shaping up topology engineering as an emerging branch of protein engineering. The synthesis of entirely protein-based molecular networks, Spy networks, is highlighted. The protocols for preparing Spy networks and applications thereof are also illustrated.We describe the operational principle, synthesis, and applications of the enzyme-DNA chimeras. These are supramolecular constructions where a DNA spring is coupled to an enzyme and introduces artificial allosteric control of the enzyme. This method is universal and can be applied to various enzymes and proteins. In addition, this method is versatile as the stresses applied by the DNA spring on the enzymes can be fine-tuned semi-continuously and thus their enzymatic activities can be modulated gradually. We give detailed protocols for the synthesis of these molecules. Summarizing our experience with different enzymes, we explain their use for fundamental studies of conformational plasticity, as well as the potential as molecular probes.Linker engineering constitutes a critical, yet frequently underestimated aspect in the construction of synthetic protein switches and sensors. Notably, systematic strategies to engineer linkers by predictive means remain largely elusive to date. This is primarily due to our insufficient understanding how the biophysical properties that underlie linker functions mediate the conformational transitions in artificially engineered protein switches and sensors. The construction of synthetic protein switches and sensors therefore heavily relies on experimental trial-and-error. Yet, methods for effectively generating linker diversity at the genetic level are scarce. Addressing this technical shortcoming, iterative functional linker cloning (iFLinkC) enables the combinatorial assembly of linker elements with functional domains from sequence verified repositories that are developed and stored in-house. The assembly process is highly scalable and given its recursive nature generates linker diversity in a combinatorial and exponential fashion based on a limited number of linker elements.Linkers play essential roles in the engineering of fusion proteins, and have been extensively demonstrated to affect protein properties such as expression level, solubility, and biological functions. For linker design and optimization, one of the key factors is the flexibility or rigidity of linkers, which describes the tendency of a linker to maintain a stable conformation when expressed, and can directly contribute to the physical distance between domains of a fusion protein. In this chapter, we discuss the design and engineering of linkers in fusion proteins, and describe a library-based method for optimization of linker flexibility. This approach is based on chimeric linkers, which are composed of both flexible and rigid (helix-forming) linker motifs. We demonstrate that the chimeric linker library capable of controlling the flexibility in a wide range can fill the gap between flexible and rigid linkers by molecular dynamics simulation and fluorescence resonance energy transfer experiments, as well as its applications in fusion protein optimization.The construction of recombinant fusion/chimeric proteins has been widely used for expression of soluble proteins and protein purification in a variety of fields of protein engineering and biotechnology. Fusion proteins are constructed by the linking of two protein domains with a peptide linker. The selection of a linker sequence is important for the construction of stable and bioactive fusion proteins. Empirically designed linkers are generally classified into two categories according to their structural features flexible linkers and rigid linkers. Selumetinib mw Rigid linkers with the α-helix-forming sequences A(EAAAK)nA (n=2-5) were first designed about two decades ago to control the distance between two protein domains and to reduce their interference. Thereafter, the helical linkers have been applied to the construction of many fusion proteins to improve expression and bioactivity. In addition, the design of fusion proteins that self-assemble into supramolecular complexes is useful for nanobiotechnology and synthetic biology. A protein that forms a self-assembling oligomer was fused by a rigid helical linker to another protein that forms another self-assembling oligomer, and the fusion protein symmetrically self-assembled into a designed protein nanoparticle or nanomaterial. Moreover, to construct chain-like polymeric nanostructures, extender protein nanobuilding blocks were designed by tandemly fusing two dimeric de novo proteins with helical or flexible linkers. The linker design of fusion proteins can affect conformation and dynamics of self-assembling nanostructures. The present review and methods focus on useful helical linkers to construct bioactive fusion proteins and protein-based nanostructures.ER/K α-helices are a subset of single alpha helical domains, which exhibit unusual stability as isolated protein secondary structures. They adopt an elongated structural conformation, while regulating the frequency of interactions between proteins or polypeptides fused to their ends. Here we review recent advances on the structure, stability and function of ER/K α-helices as linkers (ER/K linkers) in native proteins. We describe methodological considerations in the molecular cloning, protein expression and measurement of interaction strengths, using sensors incorporating ER/K linkers. We highlight biological insights obtained over the last decade by leveraging distinct biophysical features of ER/K-linked sensors. We conclude with the outlook for the use of ER/K linkers in the selective modulation of dynamic cellular interactions.Linkers are crucial to the functions of multidomain proteins as they couple functional units to encode regulation such as auto-inhibition, enzyme targeting or tuning of interaction strength. A linker changes reactions from bimolecular to unimolecular, and the equilibrium and kinetics is thus determined by the properties of the linker rather than concentrations. We present a theoretical workflow for estimating the functional consequences of tethering by a linker. We discuss how to (1) Identify flexible linkers from sequence. (2) Model the end-to-end distance distribution for a flexible linker using a worm-like chain. (3) Estimate the effective concentration of a ligand tethered by a flexible linker. (4) Calculate the decrease in binding affinity caused by auto-inhibition. (5) Calculate the expected avidity enhancement of a bivalent interaction from effective concentration. The worm-like chain modeling is available through a web application called the "Ceff calculator" (http//ceffapp.chemeslab.org), which will allow user-friendly prediction of experimentally inaccessible parameters.The use of enzymes in organic synthesis is highly appealing due their remarkably high chemo-, regio- and enantioselectivity. Nevertheless, for biosynthetic routes to be industrially useful, the enzymes must fulfill several requirements. Particularly, in case of cofactor-dependent enzymes self-sufficient systems are highly valuable. This can be achieved by fusing enzymes with complementary cofactor dependency. Such bifunctional enzymes are also relatively easy to handle, may enhance stability, and promote product intermediate channeling. However, usually the characteristics of the linker, fusing the target enzymes, are not thoroughly evaluated. A poor linker design can lead to detrimental effects on expression levels, enzyme stability and/or enzyme performance. In this chapter, the effect of the length of a glycine-rich linker was explored for the case study of ɛ-caprolactone synthesis through an alcohol dehydrogenase-cyclohexanone monooxygenase fusion system. The procedure includes cloning of linker variants, expression analysis, determination of thermostability and effect on activity and conversion levels of 15 variants of different linker sizes. The protocols can also be used for the creation of other protein-protein fusions.Peptide linkers consisting of repeats of glycine and serine residues are commonly chosen by protein engineers to introduce flexible and hydrophilic spacers between protein domains. Given the popularity of these linkers, gaining a quantitative insight in their conformational behavior is important to understand the effect on functional properties of fusion proteins, including energy transfer efficiency in luminescent sensor proteins, intramolecular domain interactions and (multivalent) binding. In this chapter, we discuss how the conformational behavior of Ser/Gly linkers can be described using random coil models, and how measuring FRET as a function of linker length can be used to obtain empirical values for the stiffness of linkers containing different Ser-to-Gly ratios. Subsequently, we show how these models and the experimentally determined linker stiffness can be used to explain and predict the functional properties of multidomain proteins, providing useful rules-of-thumb and design tools for optimal linker engineering.
Item descriptions on restaurant menus often include claims about health and other attributes, and these are much less regulated than the language on packaged food labels. This study tests whether menu items with claims have different nutritional content from items without claims.

Investigators compiled a data set of menu items, their claims, and their nutrition content using MenuStat. Data included 84,788 item-year observations at up to 96 of the top-selling restaurant chains from 2012 to 2018. Items were identified with general health, health-related ingredients, nutrient content, product sourcing, and vegan or vegetarian claims through a matching algorithm. Mixed-effects models were used to examine the effect of claims on calories, nutrients to limit (e.g., saturated fat and sodium), and other nutrients by dish types (sides, main dishes, desserts).

Most dishes with claims were lower in calories; however, items with claims were not consistently lower in other nutrients to limit (sodium, saturated fat, sugar, or trans fat). Vegan or vegetarian desserts had 128 mg (95% CI=20.9, 235.1) more sodium than desserts without this claim. Main and side dishes with claims had equivalent or higher sugar content than items without claims. Many items with claims were lower in saturated fat, especially main dishes with a nutrient content claim (-2.8 percentage points, 95% CI= -3.4, -2.2).

Items with claims were high in nutrients to limit. Additional efforts to increase transparency around excessive ingredients, such as the sodium warning labels, could be implemented by the restaurant industry.
Items with claims were high in nutrients to limit. Additional efforts to increase transparency around excessive ingredients, such as the sodium warning labels, could be implemented by the restaurant industry.
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