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 molecular architecture of these membrane proteins. Intrinsic conformational flexibility of ion channels critically governs their functions. However, the dynamics underlying gating mechanisms and modulations are obscured in the information from crystal structures. While nuclear magnetic resonance spectroscopic methods allow direct measurements of protein dynamics, they are limited by the large size of these membrane protein assemblies in detergent micelles or lipid membranes. Electron paramagnetic resonance (EPR) spectroscopy has emerged as a key biophysical tool to characterize structural dynamics of ion channels and to determine stimulus-driven conformational transition between functional states in a physiological environment. This review will provide an overview of the recent advances in the field of voltage- and ligand-gated channels and highlight some of the challenges and controversies surrounding the structural information available. It will discuss general methods used in site-directed spin labeling and EPR spectroscopy and illustrate how findings from these studies have narrowed the gap between high-resolution structures and gating mechanisms in membranes, and have thereby helped reconcile seemingly disparate models of ion channel function.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Chakrapani Sdoi
10.1016/bs.mie.2014.12.030subject
Has Abstractpub_date
2015-01-01 00:00:00pages
279-306eissn
0076-6879issn
1557-7988pii
S0076-6879(14)00144-Xjournal_volume
557pub_type
杂志文章,评审abstract::Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were d...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.11.009
更新日期:2017-01-01 00:00:00
abstract::Retinoic acid (RA), the biologically active metabolite of vitamin A, regulates a vast spectrum of biological processes, such as cell differentiation, proliferation, apoptosis, and morphogenesis. microRNAs (miRNAs) play a crucial role in regulating gene expression by binding to messenger RNA (mRNA) which leads to mRNA ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.02.009
更新日期:2020-01-01 00:00:00
abstract::Family 3 carbohydrate-binding modules (CBM3s) are among the most distinctive, diverse, and robust. CBM3s, which are numerous components of both free cellulases and cellulosomes, bind tightly to crystalline cellulose, and thus play a key role in cellulose degradation through their substrate targeting capacity. In addit...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-415931-0.00013-6
更新日期:2012-01-01 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
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.06.005
更新日期:2016-01-01 00:00:00
abstract::The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron microscopy, membrane marker enzyme analysis, binding study, EGF-dependent receptor autophosphorylation, and Western blots all demonstrate the biological activity of the ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(91)98026-3
更新日期:1991-01-01 00:00:00
abstract::Granzyme B (GrB) is the primary molecular mediator of apoptosis by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. It is a unique mammalian aspartic acid-cleaving serine protease. On T cell receptor activation, GrB is released from the CTL cytoplasmic granules by exocytosis, enters the target cells and, ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(00)22013-2
更新日期:2000-01-01 00:00:00
abstract::Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data managemen...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)11009-5
更新日期:2006-01-01 00:00:00
abstract::We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic s...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)22019-6
更新日期:2007-01-01 00:00:00
abstract::Perhaps the most important contribution of FLN is that it provides an experimental approach to relate physical changes in the protein to predicted dynamical behavior. It is clear that the sample is inhomogeneously broadened in a continuous manner, consistent with the damped motion of proteins. At the same time configu...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/s0076-6879(97)78007-8
更新日期:1997-01-01 00:00:00
abstract::Heterodimeric complexes between individual members of the R7 subfamily of regulators of G-protein signaling proteins and the Gbeta5 isoform of the heterotrimeric G-protein beta subunit family are strongly expressed in the cell nucleus in neurons and brain, as well as in the cytoplasm and plasma membrane. Native and re...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(04)90014-6
更新日期:2004-01-01 00:00:00
abstract::The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions va...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385120-8.00020-6
更新日期:2011-01-01 00:00:00
abstract::Cancer has become the leading cause of death in the developed world and has remained one of the most difficult diseases to treat. One of the difficulties in treating cancer is that conventional chemotherapies often have unacceptable toxicities toward normal cells at the doses required to kill tumor cells. Thus, the de...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/B978-0-12-416039-2.00015-X
更新日期:2012-01-01 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
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2014.10.023
更新日期:2015-01-01 00:00:00
abstract::Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin interm...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.003
更新日期:2016-01-01 00:00:00
abstract::In this article, we have described methods used to purify Rnt1p and study its biochemical properties. Rnt1p can be easily purified from bacteria as N-terminal His6-tagged protein and its activity may be monitored in vitro. Rnt1p cleaves the RNA by binding to a cleavage site followed by hydrolysis and product release. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(01)42543-2
更新日期:2001-01-01 00:00:00
abstract::Staphylococcus aureus ribonuclease III (Sa-RNase III) belongs to the enzyme family known to process double-stranded RNAs consisting of two turns of the RNA helix. Although the enzyme is thought to play a role in ribosomal RNA processing and gene regulation, the deletion of the rnc gene in S. aureus does not affect cel...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02216-7
更新日期:2008-01-01 00:00:00
abstract::Genetic modifier screens offer a powerful, indeed a uniquely powerful tool for the analysis and identification of elements capable of modulating specific cellular functions in development. Here, we describe the methodology that allowed us to explore the genetic circuitry that affects a Notch mutant phenotype caused by...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-397926-1.00016-0
更新日期:2014-01-01 00:00:00
abstract::The breakthrough discovery that double-stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence-specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 200...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)06046-0
更新日期:2006-01-01 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::This chapter presents methodologies for RNA extraction from soils coupled with competitive reverse transcription-polymerase chain reaction and capillary electrophoresis techniques. Combined, these approaches provide new capabilities to quantify gene expression in different environments and can aid our understanding of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)97019-5
更新日期:2005-01-01 00:00:00
abstract::Significant progress has been made in discovering and cloning a host of proteins, including a range of glycoproteins. The availability of their predicted amino acid sequences provides useful information, including potential N-linked glycosylation sites. However, only a limited number of protein structures have been so...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)15007-7
更新日期:2006-01-01 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
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.12.010
更新日期:2015-01-01 00:00:00
abstract::Slp4-a/granuphilin-a is a member of the synaptotagmin-like protein (Slp) family and consists of an N-terminal Slp homology domain (SHD) and C-terminal tandem C2 domains. Slp4-a is specifically localized on secretory granules in some endocrine and exocrine cells through its SHD, and it attenuates Ca(2+)-dependent dense...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03039-9
更新日期:2005-01-01 00:00:00
abstract::Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.12.037
更新日期:2019-01-01 00:00:00
abstract::Nonribosomal peptide biosynthesis is a complex enzymatic assembly responsible for producing a great diversity of bioactive peptide natural products. Due to the recurring arrangement of catalytic domains within these machineries, great interest has been shown in reengineering these pathways to produce novel, designer p...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.12.008
更新日期:2019-01-01 00:00:00
abstract::The findings in this article illustrate the complexity residing in the regulation of reversible S-glutathionylation of proteins, such as GAPDH. This is clearly reflected in the design of suitable experimental approaches designed to cope with the interaction of several redox-dependent factors. Clear interactions are de...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)48636-3
更新日期:2002-01-01 00:00:00
abstract::The human cannabinoid receptor, CB1, has been difficult to purify in a functional form, hampering structural and biophysical studies. Here, we present our approaches for obtaining pure, detergent solubilized, functional CB1. We also discuss our site-directed fluorescence labeling (SDFL) methods for identifying differe...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.06.026
更新日期:2017-01-01 00:00:00
abstract::The procedures described in this chapter have enabled us to identify and characterize monoclonal antibodies and their respective anti-idiotypes. We have developed several different types of immunoassays which afford greater flexibility to the investigator, depending on the type of antibodies desired and the availabili...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(89)78005-8
更新日期:1989-01-01 00:00:00
abstract::PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoic...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)22009-3
更新日期:2007-01-01 00:00:00
abstract::Uncoupling protein 2 (UCP2) is a member of the uncoupling protein family. It is expressed in the inner mitochondrial membrane and plays a role in the control of free radical production, oxidative damage, insulin secretion, and fatty-acid peroxide exportation. Although UCP2 expression occurs in several tissues, some of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05022-8
更新日期:2009-01-01 00:00:00