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 increase in NMR signals that complicate spectral interpretation due to signal overlap. The application of segmental isotope labeling to selected domains in multi-domain proteins can significantly facilitate spectral interpretation by reducing the number of observable signals. However, severe signal overlap may persist within individual domains that show low signal dispersion. To further reduce the number of signals and spectral complexity in such systems, we developed a procedure for selective amino acid-type labeling in individual domains of multi-domain proteins. This strategy combines efficient amino acid-type labeling amenable by cell-free protein expression with near-seamless domain ligation achievable by expressed protein ligation. By application of simple dual labeling schemes, this approach further allows residue-specific isotope labeling to position NMR-observable probes at desired sites within segments of multi-domain proteins. This chapter describes a detailed protocol for selective amino acid-type segmental labeling of multi-domain proteins and illustrates its application to a multi-domain RNA-binding protein. The applied ligation approach is further suitable for efficient ligation of unlabeled and/or uniformly labeled domains produced solely by recombinant in vivo expression.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Michel E,Allain FHdoi
10.1016/bs.mie.2015.05.028subject
Has Abstractpub_date
2015-01-01 00:00:00pages
389-422eissn
0076-6879issn
1557-7988pii
S0076-6879(15)00346-8journal_volume
565pub_type
杂志文章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
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2014.12.030
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
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2014.10.004
更新日期:2015-01-01 00:00:00
abstract::Contemporary high-throughput sequencing methods, notably RNA-Seq, permit the systematic identification and characterization of transcripts whose levels change significantly in response to altered biological states. We have described methods for the application of this methodology to a definition of the transcripts reg...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.09.005
更新日期:2018-01-01 00:00:00
abstract::RNA and DNA carry out diverse functions in biology including catalysis, splicing, gene regulation, and storage of genetic information. Interest has grown in understanding how nucleic acids perform such sophisticated functions given their limited molecular repertoire. RNA can fold into diverse shapes that often perturb...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801122-5.00009-X
更新日期:2014-01-01 00:00:00
abstract::When yeast are faced with irreparable DNA damage, they will first arrest in G2/M, via the DNA damage checkpoint pathway, but will subsequently adapt to that arrest and resume division. Here, we summarize assays that we have used to examine checkpoint adaptation. Specifically, we discuss the merits of inducing DNA dama...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)09009-9
更新日期:2006-01-01 00:00:00
abstract::The Microsealed Drug Delivery (MDD) system was developed to achieve several objectives: to maximize the bioavailability of a therapeutic agent in a target tissue; to minimize the adverse side effects of a therapeutic agent or its metabolites; to optimize the onset, rate, and duration of drug delivery; to maintain the...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(85)12036-7
更新日期:1985-01-01 00:00:00
abstract::Rab7, a member of the Rab family of small G proteins, has been shown to regulate late endocytic traffic and lysosome biogenesis, but the exact roles and the mode of actions of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins and works together with them to ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03059-4
更新日期:2005-01-01 00:00:00
abstract::Fragment-based drug discovery has emerged over the past 15 years as an effective lead discovery paradigm that is complementary to traditional high-throughput screening. The starting point for fragment-based drug discovery is the identification of low-molecular weight, typically low-affinity compounds that bind to a ta...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381274-2.00006-6
更新日期:2011-01-01 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
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)31004-5
更新日期:2007-01-01 00:00:00
abstract::ADP-ribosylation factor domain protein 1 (ARD1) is a multifunctional protein that belongs to the family of 20-kDa ARF proteins. The ARD1 gene encodes a 64-kDa protein with a structure comprising an 18-kDa ADP-ribosylation factor (ARF) domain at the C-terminus (amino acids 403-574), and a 46-kDa N-terminal domain (amin...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)04019-X
更新日期:2005-01-01 00:00:00
abstract::Genetically encoded fluorescent sensors are essential tools in modern biological research, and recent advances in fluorescent proteins (FPs) have expanded the scope of sensor design and implementation. In this review we compare different sensor platforms, including Förster resonance energy transfer (FRET) sensors, flu...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2017.01.019
更新日期:2017-01-01 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
pub_type: 杂志文章
doi:10.1016/B978-0-12-391861-1.00008-3
更新日期:2013-01-01 00:00:00
abstract::Inducible expression systems represent the founding technology for the emergence of synthetic biology in mammalian cells. The core molecules in these systems are bacterial regulator proteins that bind to or dissociate from a cognate DNA operator sequence in response to an exogenous stimulus like a small-molecule induc...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385075-1.00011-1
更新日期:2011-01-01 00:00:00
abstract::MicroRNA (miRNA) genes are transcribed into long primary transcripts (pri-miRNAs) that get processed into mature miRNAs of about 22 nt in length by two different ribonuclease (RNase) III enzymes, Drosha and Dicer. Various experimental protocols have been developed and modified for genetic and biochemical analyses for ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)27005-3
更新日期:2007-01-01 00:00:00
abstract::Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)05003-2
更新日期:2005-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)04049-8
更新日期:2005-01-01 00:00:00
abstract::This chapter reviews basic concepts of nonlinear fluorescence upconversion, a technique whose temporal resolution is essentially limited only by the pulse width of the ultrafast laser. Design aspects for upconversion spectrophotofluorometers are discussed, and a recently developed system is described. We discuss appli...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(08)03408-3
更新日期:2008-01-01 00:00:00
abstract::Glycosylphosphatidylinositol-anchored proteins can be specifically identified by several methods. PI-PLC digestion analyses, the most widely used technique, can be performed more reliably when conducted with purified protein and phase partitioning to exclude steric effects and when combined with alkaline hydrolysis to...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(95)50099-5
更新日期:1995-01-01 00:00:00
abstract::The identification of novel forms of O-linked glycosylation on epidermal growth factor and thrombospondin type 1 repeats, and their emerging functional significance, require the development of new methods for their analysis. This chapter describes detailed methods to analyze both the structure and the site of modifica...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)17008-1
更新日期:2006-01-01 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
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)82005-1
更新日期:2010-01-01 00:00:00
abstract::Prior to the development of 3D reconstruction, images were interpreted in terms of models made from simple units like ping-pong balls. Generally, people eye-balled the images and with other knowledge about its structure, such as the number of subunits, proposed models to account for the images. How was one to know if ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)81016-X
更新日期:2010-01-01 00:00:00
abstract::Because polysaccharides have very complicated chemical structures constructed by a great diversity of monosaccharide residues and glycosidic linkages, enzymatic approaches have been identified as powerful tools to precisely synthesize polysaccharides as the reactions progress in highly controlled regio- and stereoarra...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.06.009
更新日期:2019-01-01 00:00:00
abstract::Intraflagellar transport (IFT) involves the movement of large proteinaceous particles or trains along the length of ciliary and flagellar axonemal microtubules. The particles contain multiple copies of two protein complexes. As isolated from the flagellated model organism, Chlamydomonas reinhardtii, IFT A contains 6 d...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-397945-2.00010-X
更新日期:2013-01-01 00:00:00
abstract::The Escherichia coli fumarate and nitrate reductase (FNR) regulator protein is an important transcriptional regulator that controls the expression of a large regulon of more than 100 genes in response to changes in oxygen availability. FNR is active when it acquires a [4Fe-4S](2+) cluster under anaerobic conditions. T...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)37011-0
更新日期:2008-01-01 00:00:00
abstract::Posttranslational modifications (PTMs) of proteins perform crucial roles in regulating the biology of the cell. PTMs are enzymatic, covalent chemical modifications of proteins that typically occur after the translation of mRNAs. These modifications are relevant because they can potentially change a protein's physical ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(09)63040-8
更新日期:2009-01-01 00:00:00
abstract::β-arrestins (βarrs) are multifunctional proteins that interact with activated and phosphorylated G protein-coupled receptors (GPCRs) to regulate their signaling and trafficking. Understanding the intricate details of GPCR-βarr interaction continues to be a key research area in the field of GPCR biology. Bimane fluores...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.11.009
更新日期:2020-01-01 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
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.07.005
更新日期:2015-01-01 00:00:00
abstract::Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.03.017
更新日期:2017-01-01 00:00:00
abstract::To maintain cellular homeostasis, the levels of transmembrane receptors found on the plasma membrane must be tightly regulated. Endocytosis of activated receptors and the eventual degradation of these transmembrane proteins in the lysosome serve a vital role in maintaining the plasma membrane receptor levels as well a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03049-1
更新日期:2005-01-01 00:00:00
abstract::Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular ani...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.04.017
更新日期:2018-01-01 00:00:00