Catalytic mechanisms and regulation of protein kinases.

abstract：:Epitope tagged alpha 2-AR subtypes have been used to address a variety of cell biological questions, and the strategies used are readily applicable to all GPCR as well as other cell surface proteins. We have provided detailed protocols for successful utilization of the epitope-tagged receptor in the studies of protein...

journal_title：Methods in enzymology

authors： Wozniak M,Saunders C,Schramm N,Keefer JR,Limbird LE

更新日期：2002-01-01 00:00:00

abstract：:In structural proteomics, it is necessary to efficiently screen in a high-throughput manner for the presence of stable structures in proteins that can be subjected to subsequent structure determination by X-ray or NMR spectroscopy. Here we illustrate that the (1)H chemical distribution in a protein as detected by (1)H...

journal_title：Methods in enzymology

authors： Hoffmann B,Eichmüller C,Steinhauser O,Konrat R

更新日期：2005-01-01 00:00:00

abstract：:We describe a method for molecular confinement and single-fluorophore sensitive measurement in aqueous nanodroplets in oil. The sequestration of individual molecules in droplets has become a useful tool in genomics and molecular evolution. Similarly, the use of single fluorophores, or pairs of fluorophores, to study b...

journal_title：Methods in enzymology

authors： Goldner LS,Jofre AM,Tang J

更新日期：2010-01-01 00:00:00

abstract：:G-protein-coupled receptors (GPCRs) represent a major class of receptors through which a number of signals ranging from photons to large glycoprotein hormones are recognized. Human genome encodes about 800 GPCRs, yet very little structural information is available on this class of receptors. Structural studies provide...

journal_title：Methods in enzymology

authors： Ashok Y,Nanekar RT,Jaakola VP

更新日期：2013-01-01 00:00:00

abstract：:Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suite...

journal_title：Methods in enzymology

authors： Adams RL,Huston NC,Tavares RCA,Pyle AM

更新日期：2019-01-01 00:00:00

abstract：:Mutations in regulators and effectors of the Rho GTPases underlie various forms of mental retardation (MR). Among them, oligophrenin-1 (OPHN1), which encodes a Rho-GTPase activating protein, was one of the first Rho-linked MR genes identified. Upon characterization of OPHN1 in hippocampal brain slices, we obtained evi...

journal_title：Methods in enzymology

authors： Kasri NN,Govek EE,Van Aelst L

更新日期：2008-01-01 00:00:00

abstract：:For mRNAs encoding secretory and integral membrane proteins, translation initiation is thought to begin a process of mRNA localization where mRNA/ribosome/nascent chain complexes (RNCs) are trafficked from the cytosol compartment to the endoplasmic reticulum (ER). At the ER membrane, RNCs bind to a protein-conducting ...

journal_title：Methods in enzymology

authors： Stephens SB,Nicchitta CV

更新日期：2007-01-01 00:00:00

abstract：:Dynamin I is a large GTPase enzyme required in membrane constriction and fission during multiple forms of endocytosis. The first method described here is for the rapid purification of native dynamin from peripheral membrane extracts of sheep brain using ammonium sulfate precipitation and affinity purification on recom...

journal_title：Methods in enzymology

authors： Quan A,Robinson PJ

更新日期：2005-01-01 00:00:00

abstract：:Nonribosomal peptides are natural products typically of bacterial and fungal origin. These highly complex molecules display a broad spectrum of biological activities, and have been exploited for the development of immunosuppressant, antibiotic, anticancer, and other therapeutic agents. The nonribosomal peptides are as...

journal_title：Methods in enzymology

authors： Wilkinson B,Micklefield J

更新日期：2009-01-01 00:00:00

abstract：:Hexameric helicases couple the energy of ATP hydrolysis to processive movement along nucleic acids and are critical components of cells and many viruses. Molecular motion derives from ATP hydrolysis at up to six distinct catalytic centers, which is coupled to the coordinated action of translocation loops in the center...

journal_title：Methods in enzymology

authors： Thomsen ND,Berger JM

更新日期：2012-01-01 00:00:00

abstract：:As new organisms are being sequenced on a daily basis, new DNA glycosylases that recognize DNA damage can be easily identified in an effort to understand both their phylogenetics and substrate specificities. As a practical matter, existing bacterial and human homologs need to be readily available as laboratory reagent...

journal_title：Methods in enzymology

authors： Bandaru V,Blaisdell JO,Wallace SS

更新日期：2006-01-01 00:00:00

abstract：:In the near future, computational tools and methods based on the mathematical modeling of biomedically relevant networks and pathways will be necessary for the design of therapeutic strategies that fight complex multifactorial diseases. Beyond the use of pharmacokinetic and pharmacodynamic approaches, we propose here ...

journal_title：Methods in enzymology

authors： Marin-Sanguino A,Gupta SK,Voit EO,Vera J

更新日期：2011-01-01 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：:Flavoenzymes comprise a large class of proteins that carry out a diverse range of important redox chemistry. Although X-ray crystal structures of many flavoenzymes have been determined, there are still unresolved questions regarding the actual oxidation state of the flavin cofactors in these structures due to photored...

journal_title：Methods in enzymology

authors： Kerber T,Vrielink A

更新日期：2020-01-01 00:00:00

abstract：:Structure-based protein design is a powerful technique with great potential. Challenges in two areas limit performance: structure scoring and sequence-structure searching. Many of the functions used to describe the relationship between protein sequence and energy are computationally expensive to evaluate, and the spac...

journal_title：Methods in enzymology

authors： Negron C,Keating AE

更新日期：2013-01-01 00:00:00

abstract：:Biotinylated molecules are extensively employed in bioanalytics and biotechnology. The currently available assays for quantification of biotin groups suffer from low sensitivity, low accuracy, or provide highly variable responses for different biotin derivatives. We developed a competitive binding assay in which avidi...

journal_title：Methods in enzymology

authors： Oberbichler E,Wiesauer M,Schlögl E,Stangl J,Faschinger F,Knör G,Gruber HJ,Hytönen VP

更新日期：2020-01-01 00:00:00

abstract：:Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently ...

journal_title：Methods in enzymology

authors： Hoch NC,Chen ES,Tsai MD,Heierhorst J

更新日期：2017-01-01 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：:It has become increasingly clear that large RNA molecules, especially long noncoding RNAs, function in almost all gene regulatory processes (Cech & Steitz, 2014). Many large RNAs appear to be structural scaffolds for assembly of important RNA/protein complexes. However, the structures of most large cellular RNA molecu...

journal_title：Methods in enzymology

authors： Lin Y,May GE,Joel McManus C

更新日期：2015-01-01 00:00:00

abstract：:The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes. They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha. They have served a...

journal_title：Methods in enzymology

authors： Hatley ME,Gilman AG,Sunahara RK

更新日期：2002-01-01 00:00:00

abstract：:Advanced glycation is one of the major pathophysiological posttranslational modifications. Under hyperglycemic conditions or oxidative stress, proteins and DNA are nonenzymatically modified by oxidative glycation and converted to advanced glycation endproducts (AGEs), which induce the loss of protein functions or apop...

journal_title：Methods in enzymology

authors： Inagi R

更新日期：2011-01-01 00:00:00

abstract：:Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. Thus, the intracellular accumulation of ROS may not only disrupt the functions of specific tissues and organs but also ...

journal_title：Methods in enzymology

authors： Wojtala A,Bonora M,Malinska D,Pinton P,Duszynski J,Wieckowski MR

更新日期：2014-01-01 00:00:00

abstract：:Genome editing using the Cas9 endonuclease of Streptococcus pyogenes has demonstrated unprecedented efficacy and facility in a wide variety of biological systems. In zebrafish, specifically, studies have shown that Cas9 can be directed to user-defined genomic target sites via synthetic guide RNAs, enabling random or h...

journal_title：Methods in enzymology

authors： Gonzales AP,Yeh JR

更新日期：2014-01-01 00:00:00

abstract：:Live cell microscopy using fluorescent proteins and small fluorescent probes is a well-established and essential tool for cell biology; however, there is a considerable need for noninvasive techniques able to study tissue and cell dynamics without the need to introduce chemical or genetically encoded probes. Coherent ...

journal_title：Methods in enzymology

authors： Pope I,Langbein W,Borri P,Watson P

更新日期：2012-01-01 00:00:00

abstract：:Helical symmetry is commonly used for building macromolecular assemblies. Helical symmetry is naturally present in viruses and cytoskeletal filaments and also occurs during crystallization of isolated proteins, such as Ca-ATPase and the nicotinic acetyl choline receptor. Structure determination of helical assemblies b...

journal_title：Methods in enzymology

authors： Diaz R,Rice WJ,Stokes DL

更新日期：2010-01-01 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：:Sequence analysis of synthetic peptides using Edman chemistry can be very useful for the elucidation of certain types of synthetic problems, such as residue deletions and the presence of common stable derivatives, and for following the progress of the synthesis itself. However, it can also be a relatively poor techniq...

journal_title：Methods in enzymology

authors： Grant GA,Crankshaw MW,Gorka J

更新日期：1997-01-01 00:00:00

abstract：:The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes buil...

journal_title：Methods in enzymology

authors： Katyal P,Yang Y,Vinogradova O,Lin Y

更新日期：2017-01-01 00:00:00

abstract：:Gas chromatography-mass spectrometry (GC-MS) is one of the three most popular analytical platforms for metabolomics and is largely employed for the study of oncometabolism. Large volumes of data are usually generated in a GC-MS experiment, and many analytical steps are required to extract biologically relevant informa...

journal_title：Methods in enzymology

authors： Koo I,Wei X,Zhang X

更新日期：2014-01-01 00:00:00

abstract：:The elucidation of structure-affinity relationships in EF-hand proteins requires a reliable assay of divalent ion affinity. In principle, isothermal titration calorimetry (ITC) should be capable of furnishing estimates for Ca2+- and Mg2+-binding constants in these systems. And because the method yields the binding ent...

journal_title：Methods in enzymology

authors： Henzl MT

更新日期：2009-01-01 00:00:00