Abstract：
:The transmembrane (TM) anchors of cell surface proteins have been one of the 'blind spots' in structural biology because they are generally very hydrophobic, sometimes dynamic, and thus difficult targets for structural characterization. A plethora of examples show these membrane anchors are not merely anchors but can multimerize specifically to activate signaling receptors on the cell surface or to stabilize envelope proteins in viruses. Through a series of studies of the TM domains (TMDs) of immune receptors and viral membrane proteins, we have established a robust protocol for determining atomic-resolution structures of TM oligomers by NMR in bicelles that closely mimic a lipid bilayer. Our protocol overcomes hurdles typically encountered by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM) when studying small TMDs. Here, we provide the details of the protocol, covering five major technical aspects: (i) a general method for producing isotopically labeled TM or membrane-proximal (MP) protein fragments that involves expression of the protein (which is fused to TrpLE) into inclusion bodies and releasing the target protein by cyanogen bromide (CNBr) cleavage; (ii) determination of the oligomeric state of TMDs in bicelles; (iii) detection of intermolecular contacts using nuclear Overhauser effect (NOE) experiments; (iv) structure determination; and (v) paramagnetic probe titration (PPT) to characterize the membrane partition of the TM oligomers. This protocol is broadly applicable for filling structural gaps of many type I/II membrane proteins. The procedures may take 3-6 months to complete, depending on the complexity and stability of the protein sample.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Fu Q,Piai A,Chen W,Xia K,Chou JJdoi
10.1038/s41596-019-0188-9subject
Has Abstractpub_date
2019-08-01 00:00:00pages
2483-2520issue
8eissn
1754-2189issn
1750-2799pii
10.1038/s41596-019-0188-9journal_volume
14pub_type
杂志文章相关文献
Nature Protocols文献大全abstract：:Ex vivo perfusion systems offer a reliable, reproducible method for studying acute physiological responses of an organ to various environmental manipulations. Unlike in vitro culture systems, the cellular organization, compartmentalization and three-dimensional structure of ex vivo-perfused organs are maintained. Thes...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2012.144
更新日期：2013-01-01 00:00:00
abstract：:This protocol explains how to discover functional signals in genomic sequences by detecting over- or under-represented oligonucleotides (words) or spaced pairs thereof (dyads) with the Regulatory Sequence Analysis Tools (http://rsat.ulb.ac.be/rsat/). Two typical applications are presented: (i) predicting transcription...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2008.98
更新日期：2008-01-01 00:00:00
abstract：:Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been develop...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0070-1
更新日期：2018-12-01 00:00:00
abstract：:In order to meaningfully analyze common and rare genetic variants, results from genome-wide association studies (GWASs) of multiple cohorts need to be combined in a meta-analysis in order to obtain enough power. This requires all cohorts to have the same single-nucleotide polymorphisms (SNPs) in their GWASs. To this e...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.077
更新日期：2015-09-01 00:00:00
abstract：:Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.17
更新日期：2006-01-01 00:00:00
abstract：:Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple a...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.134
更新日期：2016-01-01 00:00:00
abstract：:Growing evidence indicates that RNA G-quadruplexes have important roles in various processes such as transcription, translation, regulation of telomere length, and formation of telomeric heterochromatin. Investigation of RNA G-quadruplex structures associated with biological events is therefore essential to understand...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2017.156
更新日期：2018-04-01 00:00:00
abstract：:Mouse embryonic stem cells (mESCs) are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. However, successful derivation of germline-competent embryonic stem cell lines has, until recently, been limited to a small number of inbre...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2014.030
更新日期：2014-03-01 00:00:00
abstract：:Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, thus allowing the s...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.017
更新日期：2015-02-01 00:00:00
abstract：:The use of anisotropic NMR data, such as residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs), has emerged as a powerful technique for structural characterization of organic small molecules. RDCs typically report the relative orientations of different 1H-13C bonds; RCSAs report the relati...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0091-9
更新日期：2019-01-01 00:00:00
abstract：:This protocol and the accompanying software program called LEVER (lineage editing and validation) enable quantitative automated analysis of phase-contrast time-lapse images of cultured neural stem cells. Images are captured at 5-min intervals over a period of 5-15 d as the cells proliferate and differentiate. LEVER au...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2011.422
更新日期：2011-11-17 00:00:00
abstract：:The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolutio...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2008.79
更新日期：2008-01-01 00:00:00
abstract：:Traumatic brain injury (TBI) is a major cause of mortality and morbidity. Various attempts have been made to replicate clinical TBI using animal models. The fluid-percussion model (FP) is one of the oldest and most commonly used models of experimentally induced TBI. Both central (CFP) and lateral (LFP) variations of t...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2010.112
更新日期：2010-09-01 00:00:00
abstract：:Brachypodium distachyon is emerging as a new model system for bridging research into temperate cereal crops, such as wheat and barley, and for promoting research in novel biomass grasses. Here, we provide an adapter ligation PCR protocol that allows the large-scale characterization of T-DNA insertions into the genome ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2009.32
更新日期：2009-01-01 00:00:00
abstract：:This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed detail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described pro...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.330
更新日期：2007-01-01 00:00:00
abstract：:This protocol describes a method for directing the expression of genes of interest into postmitotic neocortical neurons in vivo. Microinjection of a DNA plasmid-amphiphilic molecule mix into the neocortex followed by delivery of an ad hoc electric pulse protocol during the first few days of life in mice allows rapid, ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2015.001
更新日期：2015-01-01 00:00:00
abstract：:N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB) is a highly reactive prosthetic group for radiolabeling of proteins for use in positron emission tomography (PET). It is similar to N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB), the 'gold-standard' prosthetic group for protein (18)F-labeli...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2012.110
更新日期：2012-11-01 00:00:00
abstract：:This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.163
更新日期：2014-01-01 00:00:00
abstract：:Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammal...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-018-0118-2
更新日期：2019-03-01 00:00:00
abstract：:A procedure for the conversion of a symmetrical ketone to an enantiomerically pure lactam is described. The technique described here involves a ring-expansion reaction of a 4-substituted cyclohexanone accomplished with a chiral 1,3-azidopropanol derivative. The procedure entails first a one-step preparation of (R)-1-p...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.518
更新日期：2008-01-01 00:00:00
abstract：:The Strep-tag II is an eight-residue minimal peptide sequence (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) that exhibits intrinsic affinity toward streptavidin and can be fused to recombinant proteins in various fashions. We describe a protocol that enables quick and mild purification of corresponding Strep-tag II fusion protein...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2007.209
更新日期：2007-01-01 00:00:00
abstract：:Understanding chemical reactivity through the use of state-of-the-art computational techniques enables chemists to both predict reactivity and rationally design novel reactions. This protocol aims to provide chemists with the tools to implement a powerful and robust method for analyzing and understanding any chemical ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0265-0
更新日期：2020-02-01 00:00:00
abstract：:Rapid evaluation of microbial cell-surface carbohydrates is essential to understanding the mechanisms by which bacteria use glycans to establish pathogenic or symbiotic relationships. Microbial glycan analysis is complicated both by the vast diversity of possible carbohydrate structures and by their dynamic nature. Ba...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2006.76
更新日期：2006-01-01 00:00:00
abstract：:The development of single-molecule switching (SMS) fluorescence microscopy (also called single-molecule localization microscopy) over the last decade has enabled researchers to image cell biological structures at unprecedented resolution. Using two opposing objectives in a so-called 4Pi geometry doubles the available ...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-00428-7
更新日期：2020-12-16 00:00:00
abstract：:The glycocalyx comprises glycosylated proteins and lipids and fcorms the outermost layer of cells. It is involved in fundamental inter- and intracellular processes, including non-self-cell and self-cell recognition, cell signaling, cellular structure maintenance, and immune protection. Characterization of the glycocal...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-0350-4
更新日期：2020-08-01 00:00:00
abstract：:RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations) while opt...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2013.099
更新日期：2013-09-01 00:00:00
abstract：:Freestanding plasmonic nanoparticle (NP) superlattice sheets are novel 2D nanomaterials with tailorable properties that enable their use for broad applications in sensing, anticounterfeit measures, ionic gating, nanophotonics and flat lenses. We recently developed a robust, yet general, two-step drying-mediated approa...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-019-0200-4
更新日期：2019-09-01 00:00:00
abstract：:Systematic complex genetic interaction studies have provided insight into high-order functional redundancies and genetic network wiring of the cell. Here, we describe a method for screening and quantifying trigenic interactions from ordered arrays of yeast strains grown on agar plates as individual colonies. The proto...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/s41596-020-00456-3
更新日期：2021-01-18 00:00:00
abstract：:Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into anima...
journal_title：Nature protocols
pub_type： 杂志文章
doi：10.1038/nprot.2017.078
更新日期：2017-09-01 00:00:00
abstract：:In the version of this protocol originally published, the caption for Fig. 3 was erroneously placed with Fig. 4, and that for Fig. 4 was placed with Fig. 3. This error has been corrected in the HTML and PDF versions of the paper. ...
journal_title：Nature protocols
pub_type： 已发布勘误
doi：10.1038/s41596-018-0079-5
更新日期：2019-07-01 00:00:00
