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Methods: a Companion to Methods in Enzymology

T Gopinath, Gianluigi Veglia
The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions...
July 13, 2018: Methods: a Companion to Methods in Enzymology
Sultan Imangaliyev, Andrei Prodan, Max Nieuwdorp, Albert K Groen, Natal A W van Riel, Evgeni Levin
Mining biological information from rich "-omics" datasets is facilitated by organizing features into groups that are related to a biological phenomenon or clinical outcome. For example, microorganisms can be grouped based on a phylogenetic tree that depicts their similarities regarding genetic or physical characteristics. Here, we describe algorithms that incorporate auxiliary information in terms of groups of predictors and the relationships between them into the metagenome learning task to build intelligible models...
July 4, 2018: Methods: a Companion to Methods in Enzymology
Juan P Fernandez, Charles E Vejnar, Antonio J Giraldez, Romain Rouet, Miguel A Moreno-Mateos
The CRISPR-Cas9 system biotechnological impact has recently broadened the genome editing toolbox available to different model organisms further with the addition of new efficient CRISPR-based endonucleases. We have recently optimized CRISPR-Cpf1 (renamed Cas12a) system in zebrafish. We showed that i) in the absence of Cpf1 protein, crRNAs are unstable and degraded in vivo, and CRISPR-Cpf1 RNP complexes efficiently mutagenize the zebrafish genome; and ii) temperature modulates Cpf1 activity especially affecting AsCpf1, which experiences a reduced performance below 37°C...
June 28, 2018: Methods: a Companion to Methods in Enzymology
José A Caro, A Joshua Wand
Pressure and temperature are the two fundamental variables of thermodynamics. Temperature and chemical perturbation are central experimental tools for the exploration of macromolecular structure and dynamics. Though it has long been recognized that hydrostatic pressure offers a complementary and often unique view of macromolecular structure, stability and dynamics, it has not been employed nearly as much. For solution NMR applications the limited use of high-pressure is undoubtedly traced to difficulties of employing pressure in the context of modern multinuclear and multidimensional NMR...
June 28, 2018: Methods: a Companion to Methods in Enzymology
Scott D Gorman, Debashish Sahu, Kathleen F O'Rourke, David D Boehr
Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has helped to substantially expand the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100 kDa in size. While the toolset for studying protein structure and dynamics by NMR continues to expand, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (> 50 kDa) proteins. In this practical review, we present strategies for efficiently isotopically labeling proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis...
June 26, 2018: Methods: a Companion to Methods in Enzymology
Tripti Gupta, Gregory D Marquart, Eric J Horstick, Kathryn M Tabor, Sinisa Pajevic, Harold A Burgess
Large-scale genomic studies have recently identified genetic variants causative for major neurodevelopmental disorders, such as intellectual disability and autism. However, determining how underlying developmental processes are affected by these mutations remains a significant challenge in the field. Zebrafish is an established model system in developmental neurogenetics that may be useful in uncovering the mechanisms of these mutations. Here we describe the use of voxel-intensity, deformation field, and volume-based morphometric techniques for the systematic and unbiased analysis of gene knock-down and environmental exposure-induced phenotypes in zebrafish...
June 21, 2018: Methods: a Companion to Methods in Enzymology
Harald Pichler, Anita Emmerstorfer-Augustin
All intact cells, and their organelles, are surrounded by a ∼30 Å hydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells...
June 19, 2018: Methods: a Companion to Methods in Enzymology
Ronaldo P Panganiban, Kristin A Lambert, Man-Hsun Hsu, Zoe Laryea, Faoud T Ishmael
Chronic inflammatory diseases can be particularly challenging to diagnose and characterize, as inflammatory changes in tissue may not be present in blood. There is a crucial need to develop non-invasive biomarkers that would be useful in diagnosing disease and selecting medical therapies. For example, there are no blood tests to diagnose asthma, a common inflammatory lung disease. MicroRNA (miRNA) expression profiling in blood is emerging as a potentially sensitive and useful biomarker of many diseases. In particular, we have characterized a cost-effective PCR-based array technology to measure and profile circulating miRNAs in the plasma of patients with allergic rhinitis and asthma...
June 12, 2018: Methods: a Companion to Methods in Enzymology
Baekgyu Kim, V Narry Kim
CLIP-seq (crosslinking immunoprecipitation and sequencing) is widely used to map the binding sites of a protein of interest on the transcriptome, and generally employs UV to induce the covalent bonds between protein and RNA, which allows stringent washing. However, dsRNA is inefficiently crosslinked by UV, making it difficult to study the interactions between dsRNA binding proteins and their substrates. A dsRNA endoribonuclease DROSHA initiates the maturation of microRNA (miRNA) by cleaving primary miRNA (pri-miRNA)...
June 12, 2018: Methods: a Companion to Methods in Enzymology
Robert S H Istepanian, Turki Al-Anzi
Mobile health (m-Health) has been repeatedly called the biggest technological breakthrough of our modern times. Similarly, the concept of big data in the context of healthcare is considered one of the transformative drivers for intelligent healthcare delivery systems. In recent years, big data has become increasingly synonymous with mobile health, however key challenges of 'Big Data and mobile health', remain largely untackled. This is becoming particularly important with the continued deluge of the structured and unstructured data sets generated on daily basis from the proliferation of mobile health applications within different healthcare systems and products globally...
June 8, 2018: Methods: a Companion to Methods in Enzymology
Nila Roy Choudhury, Gracjan Michlewski
RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells...
June 8, 2018: Methods: a Companion to Methods in Enzymology
Ronghui You, Xiaodi Huang, Shanfeng Zhu
As of April 2018, UniProtKB has collected more than 115 million protein sequences. Less than 0.15% of these proteins, however, have been associated with experimental GO annotations. As such, the use of automatic protein function prediction (AFP) to reduce this huge gap becomes increasingly important. The previous studies conclude that sequence homology based methods are highly effective in AFP. In addition, mining motif, domain, and functional information from protein sequences has been found very helpful for AFP...
June 6, 2018: Methods: a Companion to Methods in Enzymology
Wen Zhang, Xiang Yue, Feng Huang, Ruoqi Liu, Yanlin Chen, Chunyang Ruan
Drug-disease associations provide important information for drug discovery and drug repositioning. Drug-disease associations can induce different effects, and the therapeutic effect attracts wide spread interest. Therefore, developing drug-disease association prediction methods is an important task, and differentiating therapeutic associations from other associations is also very important. In this paper, we formulate the known drug-disease associations as a bipartite network, and then present a novel representation for drugs and diseases based on the bipartite network and linear neighborhood similarity...
June 4, 2018: Methods: a Companion to Methods in Enzymology
Mengmeng Wu, Wanwen Zeng, Wenqiang Liu, Hairong Lv, Ting Chen, Rui Jiang
Genome-wide association studies (GWAS) have successfully discovered a number of disease-associated genetic variants in the past decade, providing an unprecedented opportunity for deciphering genetic basis of human inherited diseases. However, it is still a challenging task to extract biological knowledge from the GWAS data, due to such issues as missing heritability and weak interpretability. Indeed, the fact that the majority of discovered loci fall into noncoding regions without clear links to genes has been preventing the characterization of their functions and appealing for a sophisticated approach to bridge genetic and genomic studies...
June 3, 2018: Methods: a Companion to Methods in Enzymology
Markus Egert, Severin Weis, Sylvia Schnell
The RNA-SIP technology, introduced into molecular microbial ecology in 2002, is an elegant technique to link the structure and function of complex microbial communities, i.e. to identify microbial key-players involved in distinct degradation and assimilation processes under in-situ conditions. Due to its dependence of microbial RNA, this technique is particularly suited for environments with high numbers of very active, i.e. significantly RNA-expressing, bacteria. So far, it was mainly used in environmental studies using microbiotas from soil or water habitats...
May 30, 2018: Methods: a Companion to Methods in Enzymology
Gildas Nyame Mendendy Boussambe, Pierre Guillet, Florian Mahler, Anaïs Marconnet, Carolyn Vargas, Damien Cornut, Marine Soulié, Christine Ebel, Aline Le Roy, Anass Jawhari, Françoise Bonneté, Sandro Keller, Grégory Durand
Fluorinated surfactants have scarcely been explored for the direct extraction of proteins from membranes because fluorination is believed to abrogate detergency. However, we have recently shown that a commercially available fluorinated surfactant readily solubilizes lipid membranes, thereby suggesting that fluorination per se does not interfere with detergent activity. In this work, we developed new fluorinated surfactants that exhibit detergency in terms of both lipid-vesicle solubilization and membrane-protein extraction...
May 30, 2018: Methods: a Companion to Methods in Enzymology
Feixia Chu, Daniel T Thornton, Hieu T Nguyen
For decades, chemical cross-linking of proteins has been an established method to study protein interaction partners. The chemical cross-linking approach has recently been revived by mass spectrometric analysis of the cross-linking reaction products. Chemical cross-linking and mass spectrometric analysis (CXMS) enables the identification of residues that are close in three-dimensional (3D) space but not necessarily close in primary sequence. Therefore, this approach provides medium resolution information to guide de novo structure prediction, protein interface mapping and protein complex model building...
May 30, 2018: Methods: a Companion to Methods in Enzymology
Jung Ah Byun, Giuseppe Melacini
Elucidating the molecular mechanism of disease-related mutations (DRMs) is a critical first step towards understanding the etiology of genetic disorders. DRMs often modulate biological function by altering the free-energy landscape (FEL) of the protein associated with the mutated gene. FELs typically include ground, as well as excited, yet accessible and functionally relevant, states and DRMs may perturb both the thermodynamics and kinetics of the ground vs. excited and apo vs. holo transitions. NMR is ideally suited to map at atomic-resolution these DRM-induced FEL perturbations...
May 30, 2018: Methods: a Companion to Methods in Enzymology
Oliver K Castell, Patricia M Dijkman, Daniel N Wiseman, Alan D Goddard
The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling...
May 30, 2018: Methods: a Companion to Methods in Enzymology
Sebastien Alphonse, Ranajeet Ghose
Measurement of nuclear spin relaxation provides a powerful approach to access information about biomolecular conformational dynamics over several orders of magnitude in timescale. In several cases this knowledge in combination with spatial information from three-dimensional structures yields unique insight into protein stability and the kinetics and thermodynamics of their interactions and function. However, due to intrinsic difficulties in studying large systems using solution state nuclear magnetic resonance (NMR) approaches, until recently these measurements were limited to small-to-medium-sized systems...
May 29, 2018: Methods: a Companion to Methods in Enzymology
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