abstract：:Benzylisoquinoline alkaloids (BIAs) constitute a diverse class of plant secondary metabolites that includes the opiate analgesics morphine and codeine. Collectively, BIAs exhibit a myriad of pharmacological activities, including antimicrobial, antitussive, antispasmodic, and anticancer properties. Despite 2500 known B...

journal_title：Methods in enzymology

authors： Pyne ME,Narcross L,Fossati E,Bourgeois L,Burton E,Gold ND,Martin VJ

更新日期：2016-01-01 00:00:00

abstract：:Peptide-based strategies represent transformative approaches to fabricate functional inorganic materials under sustainable conditions by modeling the methods exploited in biology. In general, peptides with inorganic affinity and specificity have been isolated from organisms and through biocombinatorial selection techn...

journal_title：Methods in enzymology

authors： Bedford NM,Munro CJ,Knecht MR

更新日期：2016-01-01 00:00:00

abstract：:Peptides are multidentate chiral ligands capable of coordinating different metal ions. Nowadays, they can be obtained with high yield and purity, thanks to the advances on peptide/protein chemistry as well as in equipment (peptide synthesizers). Based on the identity and length of their amino acid sequences, peptides ...

journal_title：Methods in enzymology

authors： Lima LM,Iranzo O

更新日期：2016-01-01 00:00:00

abstract：:We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First...

journal_title：Methods in enzymology

authors： Hu C,Wang J

更新日期：2016-01-01 00:00:00

abstract：:Interactions between chemokines and glycosaminoglycans (GAGs) are crucial for the physiological and pathophysiological activities of chemokines. GAGs are therefore commonly designated as chemokine coreceptors which are deeply involved in the chemokine-signaling network. Studying the interaction of chemokines with GAGs...

journal_title：Methods in enzymology

authors： Kitic N,Gschwandtner M,Derler R,Gerlza T,Kungl AJ

更新日期：2016-01-01 00:00:00

abstract：:Chemokines are essential mediators of leukocyte movement in vivo. In vitro assays of leukocyte migration cannot mimic the complex interactions with other cell types and matrix needed for cells to extravasate and migrate into tissues. Therefore, in vivo strategies to study the effects and potential relevance of chemoki...

journal_title：Methods in enzymology

authors： Amaral FA,Boff D,Teixeira MM

更新日期：2016-01-01 00:00:00

abstract：:Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary ...

journal_title：Methods in enzymology

authors： Götz M,Wortmann P,Schmid S,Hugel T

更新日期：2016-01-01 00:00:00

abstract：:The kinetics of biomolecular systems can be quantified by calculating the stochastic rate constants that govern the biomolecular state vs time trajectories (i.e., state trajectories) of individual biomolecules. To do so, the experimental signal vs time trajectories (i.e., signal trajectories) obtained from observing i...

journal_title：Methods in enzymology

authors： Kinz-Thompson CD,Bailey NA,Gonzalez RL Jr

更新日期：2016-01-01 00:00:00

abstract：:Microfluidic devices have very broad applications in biological assays from simple chemotaxis assays to much more complicated 3D bioreactors. In this chapter, we describe the design and methods for performing chemotaxis assays using simple microfluidic chemotaxis chambers. With these devices, using real-time video mic...

journal_title：Methods in enzymology

authors： Sai J,Rogers M,Hockemeyer K,Wikswo JP,Richmond A

更新日期：2016-01-01 00:00:00

abstract：:The bromodomain (BrD) represents an evolutionarily conserved protein domain whose function mostly is to recognize acetylated lysine residues in histones and nuclear proteins in regulation of gene transcription in chromatin. The highly conserved BrD structure features an unusual left-handed, antiparallel four-helix bun...

journal_title：Methods in enzymology

authors： Ren C,Zeng L,Zhou MM

更新日期：2016-01-01 00:00:00

abstract：:Histone chaperones-key actors in the dynamic organization of chromatin-interact with the various histone variants to ensure their transfer in and out of chromatin. In vitro chromatin assembly assays and isolation of protein complexes using tagged histone variants provided first clues concerning their binding specifici...

journal_title：Methods in enzymology

authors： Clément C,Vassias I,Ray-Gallet D,Almouzni G

更新日期：2016-01-01 00:00:00

abstract：:Expression of fusion proteins such as MBP fusions can be used as a way to improve the solubility of the expressed protein in E. coli (Fox and Waugh, 2003; Nallamsetty et al., 2005; Nallamsetty and Waugh, 2006) and as a way to introduce an affinity purification tag. The protocol that follows was designed by the authors...

journal_title：Methods in enzymology

authors： Duong-Ly KC,Gabelli SB

更新日期：2015-01-01 00:00:00

abstract：:Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressib...

journal_title：Methods in enzymology

authors： Ramundo S,Rochaix JD

更新日期：2015-01-01 00:00:00

abstract：:Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor...

journal_title：Methods in enzymology

authors： Previs SF,Herath K,Castro-Perez J,Mahsut A,Zhou H,McLaren DG,Shah V,Rohm RJ,Stout SJ,Zhong W,Wang SP,Johns DG,Hubbard BK,Cleary MA,Roddy TP

更新日期：2015-01-01 00:00:00

abstract：:Genetically encodable RNA devices that directly detect small molecules in the cellular environment are of increasing interest for a variety of applications including live cell imaging and synthetic biology. Riboswitches are naturally occurring sensors of intracellular metabolites, primarily found in the bacterial mRNA...

journal_title：Methods in enzymology

authors： Trausch JJ,Batey RT

更新日期：2015-01-01 00:00:00

abstract：:Riboswitches present a ubiquitous genetic regulatory mechanism for prokaryotes and have been found in HIV1, fungi, plants, and even H. sapiens. We present an overview of approaches to predict riboswitch aptamers and, more generally, RNA conformational switches. ...

journal_title：Methods in enzymology

authors： Clote P

更新日期：2015-01-01 00:00:00

abstract：:Reliable modeling of RNA tertiary structures is key to both understanding these structures' roles in complex biological machines and to eventually facilitating their design for molecular computing and robotics. In recent years, a concerted effort to improve computational prediction of RNA structure through the RNA-Puz...

journal_title：Methods in enzymology

authors： Cheng CY,Chou FC,Das R

更新日期：2015-01-01 00:00:00

abstract：:Sedimentation velocity experiments measure the transport of molecules in solution under centrifugal force. Here, we describe a method for monitoring the sedimentation of very large biological molecular assemblies using the interference optical systems of the analytical ultracentrifuge. The mass, partial-specific volum...

journal_title：Methods in enzymology

authors： Rogge RA,Hansen JC

更新日期：2015-01-01 00:00:00

abstract：:ClpB belongs to the Hsp100 family of ring-forming heat-shock proteins involved in degradation of unfolded/misfolded proteins and in reactivation of protein aggregates. ClpB monomers reversibly associate to form the hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stres...

journal_title：Methods in enzymology

authors： Alfonso C,del Castillo U,Martín I,Muga A,Rivas G

更新日期：2015-01-01 00:00:00

abstract：:Synthetic hydrogen sulfide (H2S) donors are useful research tools as well as potential therapeutic agents. In this chapter, we report the detailed protocols for the synthesis and evaluation of a series of phosphorodithioate-based H2S donors. Fluorescence assays were used to determine H2S release from the donors in bot...

journal_title：Methods in enzymology

authors： Park CM,Xian M

更新日期：2015-01-01 00:00:00

abstract：:EPR long-range distance measurements on spin-labeled macromolecules have recently become a popular tool in structural biology. The method can be used to obtain coarse-grained structures of biomolecules, to track conformational changes and dynamics, to dock macromolecular complexes, or to localize spin centers within m...

journal_title：Methods in enzymology

authors： Hagelueken G,Abdullin D,Schiemann O

更新日期：2015-01-01 00:00:00

abstract：:Pulsed EPR methods for the study of drug binding to heme-thiolate enzymes such as cytochrome P450 and nitric oxide synthase are discussed. HYSCORE and ENDOR methods to measure (1)H of axial ligands of the heme group are described with illustrations of water serving as the axial ligand in the drug-free enzyme and ligan...

journal_title：Methods in enzymology

authors： Cruce AA,Lockart M,Bowman MK

更新日期：2015-01-01 00:00:00

abstract：:An overview of resolution enhancement of conventional, field-swept, continuous-wave electron paramagnetic resonance spectra using Fourier transform-based deconvolution methods is presented. Basic steps that are involved in resolution enhancement of calculated spectra using an implementation based on complex discrete F...

journal_title：Methods in enzymology

authors： Reed GH,Poyner RR

更新日期：2015-01-01 00:00:00

abstract：:Circadian clocks are present in most cells and are essential for maintenance of daily rhythms in physiology, mood, and cognition. Thus, not only neurons of the central circadian pacemaker but also many other peripheral tissues possess the same functional and self-sustained circadian clocks. Surprisingly, however, thei...

journal_title：Methods in enzymology

authors： Gaspar L,Brown SA

更新日期：2015-01-01 00:00:00

abstract：:Circadian clocks control daily rhythms in physiology and behavior across all phyla. These rhythms are intrinsic to individual cells that must synchronize to their environment and to each other to anticipate daily events. Recent advances in recording from large numbers of cells for many circadian cycles have enabled re...

journal_title：Methods in enzymology

authors： Herzog ED,Kiss IZ,Mazuski C

更新日期：2015-01-01 00:00:00

abstract：:The technique of site-directed spin labeling (SDSL) provides unique information on biomolecules by monitoring the behavior of a stable radical tag (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. In this chapter, we describe an approach in which SDSL is integrated with computational modelin...

journal_title：Methods in enzymology

authors： Tangprasertchai NS,Zhang X,Ding Y,Tham K,Rohs R,Haworth IS,Qin PZ

更新日期：2015-01-01 00:00:00

abstract：:Pulsed electron double resonance technique, also known as double electron-electron resonance, jointly with site-directed spin labeling (SDSL) have been used extensively for studying structures and structural change. During the last decades, significant enhancements have been made by optimization of the experimental pr...

journal_title：Methods in enzymology

authors： El Mkami H,Norman DG

更新日期：2015-01-01 00:00:00

abstract：:The major facilitator superfamily (MFS) is a diverse group of secondary transporters with members found in all kingdoms of life. The paradigm for MFS is the lactose permease (LacY) of Escherichia coli, which has been the test bed for the development of many methods applied for the analysis of transport proteins. X-ray...

journal_title：Methods in enzymology

authors： Madej MG

更新日期：2015-01-01 00:00:00

abstract：:Ion channels open and close in response to diverse stimuli, and the molecular events underlying these processes are extensively modulated by ligands of both endogenous and exogenous origin. In the past decade, high-resolution structures of several channel types have been solved, providing unprecedented details of the ...

journal_title：Methods in enzymology

authors： Chakrapani S

更新日期：2015-01-01 00:00:00

abstract：:Membrane proteins (MPs) are insoluble in aqueous media as a result of incompatibility between the hydrophilic property of the solvent molecules and the hydrophobic nature of MP surfaces, normally associated with lipid membranes. Amphipathic compounds are necessary for extraction of these macromolecules from the native...

journal_title：Methods in enzymology

authors： Sadaf A,Cho KH,Byrne B,Chae PS

更新日期：2015-01-01 00:00:00

abstract：:The steady technical advances of nuclear magnetic resonance (NMR) over the past decades enabled a significant increase in the molecular size of protein particles that can be subjected to a structural and functional characterization in solution. The larger molecular weight of such proteins is accompanied with an increa...

journal_title：Methods in enzymology

authors： Michel E,Allain FH

更新日期：2015-01-01 00:00:00

abstract：:Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media for...

journal_title：Methods in enzymology

authors： O'Neill H,Shah R,Evans BR,He J,Pingali SV,Chundawat SP,Jones AD,Langan P,Davison BH,Urban V

更新日期：2015-01-01 00:00:00

abstract：:In this chapter, we describe a set of complementary techniques that we use to study the activation of rhodopsin, a G protein-coupled receptor (GPCR), and its functional interactions with G protein and arrestin. The protein reagents used for these studies come from native disc membranes or heterologous expression, and ...

journal_title：Methods in enzymology

authors： Sommer ME,Elgeti M,Hildebrand PW,Szczepek M,Hofmann KP,Scheerer P

更新日期：2015-01-01 00:00:00

abstract：:Cell-free (CF) protein expression has emerged as one of the most efficient production platforms for membrane proteins. Central bottlenecks prevalent in conventional cell-based expression systems such as mistargeting, inclusion body formation, degradation as well as product toxicity can be addressed by taking advantage...

journal_title：Methods in enzymology

authors： Henrich E,Dötsch V,Bernhard F

更新日期：2015-01-01 00:00:00

abstract：:This protocol describes the expression and analysis of membrane proteins produced in yeast, as illustrated with Yarrowia lipolytica and Pichia pastoris. Step by step, we explain how to generate a yeast strain expressing the membrane protein of interest, how to prepare a membrane protein sample from yeast, and how to a...

journal_title：Methods in enzymology

authors： Claes K,Guerfal M,Callewaert N

更新日期：2015-01-01 00:00:00

abstract：:RNA modifications, with over one hundred known so far, are commonly proposed to fine-tune the structure and function of RNA. While modifications in rRNA and tRNA are used to modulate RNA folding and decoding properties, little is known about the function of internal modifications in mRNA/lncRNA, which includes N(6)-me...

journal_title：Methods in enzymology

authors： Liu N,Pan T

更新日期：2015-01-01 00:00:00

abstract：:Structural approaches have provided insight into the mechanisms of circadian clock oscillators. This review focuses upon the myriad structural methods that have been applied to the molecular architecture of cyanobacterial circadian proteins, their interactions with each other, and the mechanism of the KaiABC posttrans...

journal_title：Methods in enzymology

authors： Egli M

更新日期：2015-01-01 00:00:00

abstract：:The circadian clock exists to synchronize inner physiology with the external world, allowing life to anticipate and adapt to the continual changes that occur in an organism's environment. The clock architecture is highly conserved, present in almost all major branches of life. Within eukaryotes, the filamentous fungus...

journal_title：Methods in enzymology

authors： Hurley J,Loros JJ,Dunlap JC

更新日期：2015-01-01 00:00:00

abstract：:Hydrogen sulfide is an endogenous gaseous mediator that plays important roles in many physiological processes in microbes, plants, and animals. This chapter focuses on the important roles of hydrogen sulfide in protecting tissues against injury, promoting the repair of damage, and downregulating the inflammatory respo...

journal_title：Methods in enzymology

authors： Gemici B,Wallace JL

更新日期：2015-01-01 00:00:00

abstract：:RNA-binding proteins (RBPs) coordinate post-transcriptional control of gene expression, often through sequence-specific recognition of primary transcripts or mature messenger RNAs. Hundreds of RBPs are encoded in the human genome, most with undefined or incompletely defined biological roles. Understanding the function...

journal_title：Methods in enzymology

authors： Lambert NJ,Robertson AD,Burge CB

更新日期：2015-01-01 00:00:00