19,960 results match your criteria Methods in enzymology[Journal]


Preface.

Authors:
Scott Bailey

Methods Enzymol 2019 ;616:xvii-xviii

Johns Hopkins University.

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http://dx.doi.org/10.1016/S0076-6879(19)30017-5DOI Listing
January 2019
1 Read

Probing Cascade complex composition and stability using native mass spectrometry techniques.

Methods Enzymol 2019 28;616:87-116. Epub 2018 Dec 28.

Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, United States. Electronic address:

Adaptive prokaryotic immune systems rely on clustered regularly interspaced short palindromic repeats and their associated genes to provide the components necessary to clear infection by foreign genetic elements. These immune systems are based on highly specific nucleases that bind DNA or RNA and, upon sequence recognition, degrade the bound nucleic acid. Because of their specificity, CRISPR-Cas systems are being co-opted to edit genes in eukaryotic cells. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183043
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http://dx.doi.org/10.1016/bs.mie.2018.10.018DOI Listing
December 2018
2 Reads

Fluorescence-based methods for measuring target interference by CRISPR-Cas systems.

Methods Enzymol 2019 21;616:61-85. Epub 2018 Dec 21.

Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, United States. Electronic address:

Type I, II, and V CRISPR-Cas systems are RNA-guided dsDNA targeting defense mechanisms found in bacteria and archaea. During CRISPR interference, Cas effectors use CRISPR-derived RNAs (crRNAs) as guides to bind complementary sequences in foreign dsDNA, leading to the cleavage and destruction of the DNA target. Mutations within the target or in the protospacer adjacent motif can reduce the level of CRISPR interference, although the level of defect is dependent on the type and position of the mutation, as well as the guide sequence of the crRNA. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183044
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http://dx.doi.org/10.1016/bs.mie.2018.10.027DOI Listing
December 2018
3 Reads

Sortase-mediated fluorescent labeling of CRISPR complexes.

Methods Enzymol 2019 17;616:43-59. Epub 2018 Dec 17.

Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States; Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, United States. Electronic address:

Fluorescent labeling of proteins is a critical requirement for single-molecule imaging studies. Many protein labeling strategies require harsh conditions or large epitopes that can inactivate the target protein, either by decreasing the protein's enzymatic activity or by blocking protein-protein interactions. Here, we provide a detailed protocol to efficiently label CRISPR-Cas complexes with a small fluorescent peptide via sortase-mediated transpeptidation. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183045
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http://dx.doi.org/10.1016/bs.mie.2018.10.031DOI Listing
December 2018
7 Reads

Reconstitution of CRISPR adaptation in vitro and its detection by PCR.

Methods Enzymol 2019 12;616:411-433. Epub 2019 Jan 12.

Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Electronic address:

CRISPR adaptation is the initial step in CRISPR-Cas immunity and involves the acquisition of foreign invading DNA. Acquisition is facilitated by the almost universally conserved proteins Cas1 and Cas2, which form an adaptation complex. The Cas1-Cas2 complex binds fragments of invading DNA, completes final processing, and catalyzes integration into specific host loci called CRISPR arrays. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.024DOI Listing
January 2019
1 Read

CRISPR-Cas10 assisted editing of virulent staphylococcal phages.

Methods Enzymol 2019 17;616:385-409. Epub 2018 Dec 17.

Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, United States. Electronic address:

Phages are the most abundant entities in the biosphere and profoundly impact the bacterial populations within and around us. They attach to a specific host, inject their DNA, hijack the host's cellular processes, and replicate exponentially while destroying the host. Historically, phages have been exploited as powerful antimicrobials, and phage-derived proteins have constituted the basis for numerous biotechnological applications. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.023DOI Listing
December 2018
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Adapting dCas9-APEX2 for subnuclear proteomic profiling.

Methods Enzymol 2019 10;616:365-383. Epub 2018 Dec 10.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, United States. Electronic address:

Genome organization and subnuclear protein localization are essential for normal cellular function and have been implicated in the control of gene expression, DNA replication, and genomic stability. The coupling of chromatin conformation capture (3C), chromatin immunoprecipitation and sequencing, and related techniques have continuously improved our understanding of genome architecture. To profile site-specifically DNA-associated proteins in a high-throughput and unbiased manner, the RNA-programmable CRISPR-Cas9 platform has recently been combined with an enzymatic labeling system to allow proteomic landscapes at repetitive and nonrepetitive loci to be defined with unprecedented ease and resolution. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183044
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http://dx.doi.org/10.1016/bs.mie.2018.10.030DOI Listing
December 2018
2 Reads

CRISPR-Cas molecular beacons as tool for studies of assembly of CRISPR-Cas effector complexes and their interactions with DNA.

Methods Enzymol 2019 1;616:337-363. Epub 2018 Dec 1.

Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers, State University of New Jersey, Piscataway, NJ, United States; Center for Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo, Russia; Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, Russia. Electronic address:

CRISPR-Cas systems protect prokaryotic cells from invading phages and plasmids by recognizing and cleaving foreign nucleic acid sequences specified by CRISPR RNA spacer sequences. Several CRISPR-Cas systems have been widely used as tool for genetic engineering. In DNA-targeting CRISPR-Cas nucleoprotein effector complexes, the CRISPR RNA forms a hybrid with the complementary strand of foreign DNA, displacing the noncomplementary strand to form an R-loop. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.026DOI Listing
December 2018

Single-molecule FRET studies of Cas9 endonuclease.

Methods Enzymol 2019 17;616:313-335. Epub 2018 Dec 17.

Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Delft, The Netherlands. Electronic address:

Since its discovery, the CRISPR-Cas9 system has been in the center of attention for its promising applications in genome editing. However, in order to apply this system successfully in genetic engineering, all aspects of its molecular mechanism have to be well understood. One of the best ways to investigate the intricacies of the molecular mechanism is single-molecule studies as they allow real-time observation of the kinetic processes and provide high spatiotemporal resolution. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.11.002DOI Listing
December 2018
1 Read

Kinetic characterization of Cas9 enzymes.

Methods Enzymol 2019 14;616:289-311. Epub 2019 Jan 14.

Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, United States; Institute for Molecular and Cellular Biology, University of Texas at Austin, Austin, TX, United States. Electronic address:

Bacterial adaptive immune systems employ clustered regularly interspaced short palindromic repeats (CRISPR) along with their CRISPR-associated genes (Cas) to form CRISPR RNA (crRNA)-guided surveillance complexes, which target foreign nucleic acids for destruction. Cas9 is unique in that it is composed of a single polypeptide that utilizes both a crRNA and a trans-activating crRNA (tracrRNA) or a single guide RNA to create double-stranded breaks in sequences complementary to the RNA via the HNH and RuvC nuclease domains. Cas9 has become a revolutionary tool for gene-editing applications. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.022DOI Listing
January 2019

Reconstitution and biochemical characterization of ribonucleoprotein complexes in Type I-E CRISPR-Cas systems.

Methods Enzymol 2019 17;616:27-41. Epub 2018 Dec 17.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States. Electronic address:

Type I CRISPR-Cas, the most prevalent CRISPR system, features a sequential target searching and degradation process. First, the multisubunit surveillance complex Cascade recognizes the matching dsDNA target flanked by protospacer adjacent motif (PAM), promotes the heteroduplex formation between CRISPR RNA (crRNA) and the target strand (TS) DNA, and displaces the nontarget strand (NTS) DNA, resulting in R-loop formation. The helicase-nuclease fusion enzyme Cas3 is then specifically recruited to Cascade/R-loop, nicks, and processively degrades the DNA target. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.11.003DOI Listing
December 2018
1 Read

Directed evolution studies of a thermophilic Type II-C Cas9.

Methods Enzymol 2019 28;616:265-288. Epub 2018 Dec 28.

Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, United States; Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL, United States. Electronic address:

Though making up nearly half of the known CRISPR-Cas9 family of enzymes, the Type II-C CRISPR-Cas9 has been underexplored for their molecular mechanisms and potential in safe gene editing applications. In comparison with the more popular Type II-A CRISPR-Cas9, the Type II-C enzymes are generally smaller in size and utilize longer base pairing in identification of their DNA substrates. These characteristics suggest easier portability and potentially less off-targets for Type II-C in gene editing applications. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.029DOI Listing
December 2018
2 Reads

Preparation and electroporation of Cas12a/Cpf1-guide RNA complexes for introducing large gene deletions in mouse embryonic stem cells.

Methods Enzymol 2019 17;616:241-263. Epub 2019 Jan 17.

Department of Biochemistry, University of Zurich, Zurich, Switzerland. Electronic address:

CRISPR-Cas12a is a bacterial RNA-guided deoxyribonuclease that has been adopted for genetic engineering in a broad variety of organisms. Here, we describe protocols for the preparation and application of AsCas12a-guide RNA ribonucleoprotein (RNP) complexes for engineering gene deletions in mouse embryonic stem (ES) cells. We provide detailed protocols for purification of an NLS-containing AsCas12a-eGFP fusion protein, design of guide RNAs, assembly of RNP complexes, and transfection of mouse ES cells by electroporation. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.028DOI Listing
January 2019

A pipeline for characterization of novel Cas9 orthologs.

Methods Enzymol 2019 27;616:219-240. Epub 2018 Dec 27.

Institute of Biotechnology, Vilnius University, Vilnius, Lithuania. Electronic address:

In recent years, Cas9 has revolutionized the genome-editing field and enabled a broad range of applications from basic biology to biotechnology and medicine. Cas9 specificity is dictated by base pairing of the guide RNA to the complementary DNA strand, however to initiate hybridization, a short protospacer adjacent motif (PAM) sequence is required in the vicinity of the target sequence. The PAM is recognized by the Cas9 protein and varies between Cas9s. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.021DOI Listing
December 2018

Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems.

Methods Enzymol 2019 12;616:191-218. Epub 2019 Jan 12.

Biomedical Sciences Research Complex, School of Biology, University of St Andrews, St Andrews, Fife, United Kingdom. Electronic address:

Type III CRISPR effector complexes utilize a bound CRISPR RNA (crRNA) to detect the presence of RNA from invading mobile genetic elements in the cell. This RNA binding results in the activation of two enzymatic domains of the Cas10 subunit-the HD nuclease domain, which degrades DNA, and PALM/cyclase domain. The latter synthesizes cyclic oligoadenylate (cOA) molecules by polymerizing ATP, and cOA acts as a second messenger in the cell, switching on the antiviral response by activating host ribonucleases and other proteins. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.020DOI Listing
January 2019

In vitro assembly of thermostable Csm complex in CRISPR-Cas type III/A system.

Methods Enzymol 2019 12;616:173-189. Epub 2019 Jan 12.

Korea Research Institute of Bioscience & Biotechnology (KRIBB), Daejeon, South Korea; Department of Bioanalytical Science, University of Science and Technology, Daejeon, South Korea. Electronic address:

The CRISPR-Cas system is the prokaryotic immune response that destroys invading foreign nucleic acids. Based on the architecture and distinct mechanism of targeting, the CRISPR-Cas system is classified into six types (I-VI). The Csm complex belongs to the type III system and consists of five subunits (Cas10 and Csm2-5) and a crRNA. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.025DOI Listing
January 2019

Live-cell single-particle tracking photoactivated localization microscopy of Cascade-mediated DNA surveillance.

Methods Enzymol 2019 12;616:133-171. Epub 2019 Jan 12.

LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany; Prokaryotic Small RNA Biology Group, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany. Electronic address:

Type I CRISPR-Cas systems utilize small CRISPR RNA (crRNA) molecules to scan DNA strands for target regions. Different crRNAs are bound by several CRISPR-associated (Cas) protein subunits that form the stable ribonucleoprotein complex Cascade. The Cascade-mediated DNA surveillance process requires a sufficient degree of base-complementarity between crRNA and target sequences and relies on the recognition of small DNA motifs, termed protospacer adjacent motifs. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.11.001DOI Listing
January 2019

High-throughput determination of in vivo DNA sequence preferences for Cas protein binding using Library-ChIP.

Authors:
Joseph T Wade

Methods Enzymol 2019 17;616:117-132. Epub 2018 Dec 17.

Wadsworth Center, New York State Department of Health, Albany, NY, United States; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY, United States. Electronic address:

The specificity of CRISPR-Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR-Cas systems for biotechnology applications. Moreover, the specificity of CRISPR-Cas systems plays an important role in prokaryote evolution due to its role in distinguishing self from nonself. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.019DOI Listing
December 2018

Predicting and visualizing features of CRISPR-Cas systems.

Methods Enzymol 2019 19;616:1-25. Epub 2018 Dec 19.

Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, United States; Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, NC, United States. Electronic address:

Pervasive application of CRISPR-Cas systems in genome editing has prompted an increase in both interest and necessity to further elucidate existing systems as well as discover putative novel systems. The ubiquity and power of current computational platforms have made in silico approaches to CRISPR-Cas identification and characterization accessible to a wider audience and increasingly amenable for processing extensive data sets. Here, we describe in silico methods for predicting and visualizing notable features of CRISPR-Cas systems, including Cas domain determination, CRISPR array visualization, and inference of the protospacer-adjacent motif. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.016DOI Listing
December 2018

Preface.

Authors:
A Joshua Wand

Methods Enzymol 2019 ;615:xv-xvii

Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1016/S0076-6879(18)30558-5DOI Listing
January 2019

Characterizing Protein Hydration Dynamics Using Solution NMR Spectroscopy.

Methods Enzymol 2019 4;615:77-101. Epub 2018 Dec 4.

Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Protein hydration is a critical aspect of protein stability, folding, and function and yet remains difficult to characterize experimentally. Solution NMR offers a route to a site-resolved view of the dynamics of protein-water interactions through the nuclear Overhauser effects between hydration water and the protein in the laboratory (NOE) and rotating (ROE) frames of reference. However, several artifacts and limitations including contaminating contributions from bulk water potentially plague this general approach and the corruption of measured NOEs and ROEs by hydrogen exchange-relayed magnetization. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358200PMC
December 2018
2 Reads

Applications of Dissolution-DNP for NMR Screening.

Methods Enzymol 2019 4;615:501-526. Epub 2018 Dec 4.

Chemistry Department, Texas A&M University, College Station, TX, United States. Electronic address:

Experimental screening for protein-ligand interactions is a central task in drug discovery. Nuclear magnetic resonance (NMR) spectroscopy enables the determination of binding affinities, as well as the measurement of structural and dynamic parameters governing the interaction. With traditional liquid-state NMR relying on a nuclear spin polarization on the order of 10, hyperpolarization methods such as dissolution dynamic nuclear polarization (D-DNP) can increase signals by several orders of magnitude. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.016DOI Listing
December 2018
2 Reads

Protein-Small Molecule Interactions by WaterLOGSY.

Methods Enzymol 2019 20;615:477-500. Epub 2018 Sep 20.

School of Chemical Sciences, The University of Auckland, Auckland, New Zealand. Electronic address:

WaterLOGSY is a ligand-observed NMR method that is widely used for the studies of protein-small molecule interactions. The basis of waterLOGSY relies on the transfer of magnetization between water molecules, proteins, and small molecules via the nuclear Overhauser effect and chemical exchange. WaterLOGSY is used extensively for the screening of protein ligands, as it is a robust, relatively high-throughput, and reliable method to identify small molecules that bind proteins with a binding affinity (K) in the μM to mM region. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.020DOI Listing
September 2018

An ELISA-Based Screening Platform for Ligand-Receptor Discovery.

Methods Enzymol 2019 28;615:453-475. Epub 2018 Dec 28.

Child Health Institute of New Jersey, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States; Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States; Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, New Brunswick, NJ, United States. Electronic address:

Cell surface molecules are important for development and function of multicellular organisms. Although several methods are available to identify ligand-receptor pairs, ELISA-based methods are particularly amenable to high-throughput screens. ELISA-based methods have high sensitivity and low false-positive rates for detecting protein-protein interaction (PPI) complexes. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.10.001DOI Listing
December 2018

Reverse Micelle Encapsulation of Proteins for NMR Spectroscopy.

Methods Enzymol 2019 10;615:43-75. Epub 2018 Dec 10.

Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Reverse micelle (RM) encapsulation of proteins for NMR spectroscopy has many advantages over standard NMR methods such as enhanced tumbling and improved sensitivity. It has opened many otherwise difficult lines of investigation including the study of membrane-associated proteins, large soluble proteins, unstable protein states, and the study of protein surface hydration dynamics. Recent technological developments have extended the ability of RM encapsulation with high structural fidelity for nearly all proteins and thereby allow high-quality state-of-the-art NMR spectroscopy. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.032DOI Listing
December 2018
1 Read

STD NMR as a Technique for Ligand Screening and Structural Studies.

Methods Enzymol 2019 14;615:423-451. Epub 2018 Sep 14.

School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, United Kingdom. Electronic address:

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule. Such information is invaluable both in the drug discovery sector and in understanding fundamental biological interactions. Recently, powerful methods have been developed to extract a greater wealth of information from the STD NMR experiment, including ligand binding epitopes, dissociation constant determination, and mapping of binding site properties. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.018DOI Listing
September 2018

Identification of Unknown Metabolomics Mixture Compounds by Combining NMR, MS, and Cheminformatics.

Methods Enzymol 2019 7;615:407-422. Epub 2018 Dec 7.

Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, United States; Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United States; Campus Chemical Instrument Center (CCIC), The Ohio State University, Columbus, OH, United States; Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, United States. Electronic address:

Metabolomics aims at the comprehensive identification of metabolites in complex mixtures to characterize the state of a biological system and elucidate their roles in biochemical pathways. For many biological samples, a large number of spectral features observed by NMR spectroscopy and mass spectrometry (MS) belong to unknowns, i.e. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.003DOI Listing
December 2018
1 Read

DNP-Assisted NMR Investigation of Proteins at Endogenous Levels in Cellular Milieu.

Methods Enzymol 2019 18;615:373-406. Epub 2018 Sep 18.

UT Southwestern Medical Center, Dallas, TX, United States. Electronic address:

Structural investigations of biomolecules are typically confined to in vitro systems under extremely limited conditions. These investigations yield invaluable insights, but such experiments cannot capture important structural features imposed by cellular environments. Structural studies of proteins in their native contexts are not only possible using state-of-the-art sensitivity-enhanced (dynamic nuclear polarization, DNP) solid-state nuclear magnetic resonance (NMR) techniques, but these studies also demonstrate that the cellular context can and does have a dramatic influence on protein structure. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.023DOI Listing
September 2018

Solid-State NMR Spectroscopy of RNA.

Methods Enzymol 2019 7;615:333-371. Epub 2018 Dec 7.

Centre for Biomolecular Drug Research (BMWZ) and Institute of Organic Chemistry, Leibniz University Hannover, Hannover, Germany; Helmholtz Centre for Infection Research, Group of NMR-based Structural Chemistry, Braunschweig, Germany. Electronic address:

RNA structure is essential to understand RNA function and regulation in cellular processes. RNA acts either in isolation or as part of a complex with proteins. Both isolated and protein-complexed RNA represent a challenge for structural biology, due to the complexity of its conformational space and intrinsic dynamics. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.029DOI Listing
December 2018
1 Read

NMR Methods for Characterizing the Basic Side Chains of Proteins: Electrostatic Interactions, Hydrogen Bonds, and Conformational Dynamics.

Methods Enzymol 2019 27;615:285-332. Epub 2018 Sep 27.

Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, United States. Electronic address:

NMR spectroscopy is a powerful tool for studying protein dynamics. Conventionally, NMR studies on protein dynamics have probed motions of protein backbone NH, side-chain aromatic, and CH groups. Recently, there has been remarkable progress in NMR methodologies that can characterize motions of cationic groups in protein side chains. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.017DOI Listing
September 2018

Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation.

Methods Enzymol 2019 8;615:237-284. Epub 2018 Dec 8.

Johnson Research Foundation and Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States; Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

Recent studies suggest that the fast timescale motion of methyl-bearing side chains may play an important role in mediating protein activity. These motions have been shown to encapsulate the residual conformational entropy of the folded state that can potentially contribute to the energetics of protein function. Here, we provide an overview of how to characterize these motions using nuclear magnetic resonance (NMR) spin relaxation methods. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364297PMC
December 2018

Chemical Exchange.

Methods Enzymol 2019 4;615:177-236. Epub 2018 Dec 4.

Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, United States.

The phenomenon of chemical or conformational exchange in NMR spectroscopy has enabled detailed characterization of time-dependent aspects of biomolecular function, including folding, molecular recognition, allostery, and catalysis, on timescales from microsecond to second. Importantly, NMR methods based on a variety of spin relaxation parameters have been developed that provide quantitative information on interconversion kinetics, thermodynamic properties, and structural features of molecular states populated to a fraction of a percent at equilibrium and otherwise unobservable by other NMR approaches. The ongoing development of more sophisticated experimental techniques and the necessity to apply these methods to larger and more complex molecular systems engenders a corresponding need for theoretical advances describing such techniques and facilitating data analysis in applications. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.028DOI Listing
December 2018

Overhauser Dynamic Nuclear Polarization for the Study of Hydration Dynamics, Explained.

Methods Enzymol 2019 11;615:131-175. Epub 2018 Dec 11.

Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, United States; Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA, United States.

We outline the physical properties of hydration water that are captured by Overhauser Dynamic Nuclear Polarization (ODNP) relaxometry and explore the insights that ODNP yields about the water and the surface that this water is coupled to. As ODNP relies on the pairwise cross-relaxation between the electron spin of a spin probe and a proton nuclear spin of water, it captures the dynamics of single-particle diffusion of an ensemble of water molecules moving near the spin probe. ODNP principally utilizes the same physics as other nuclear magnetic resonance (NMR) relaxometry (i. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.024DOI Listing
December 2018

Understanding Protein Function Through an Ensemble Description: Characterization of Functional States by F NMR.

Methods Enzymol 2019 8;615:103-130. Epub 2018 Nov 8.

Department of Chemistry, University of Toronto, UTM, Mississauga, ON, Canada; Department of Biochemistry, University of Toronto, Toronto, ON, Canada. Electronic address:

Protein function is a consequence of a complex and dynamic equilibrium between allosterically coupled functional states. However, it is often difficult to distinguish the representative members of an ensemble by spectroscopic means. F NMR is particularly useful in this regard owing to the sensitivity of its chemical shift to subtle differences in environment. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.029DOI Listing
November 2018

Companion Simulations and Modeling to NMR-Based Dynamical Studies of Proteins.

Authors:
Kim A Sharp

Methods Enzymol 2019 8;615:1-41. Epub 2018 Oct 8.

Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States. Electronic address:

NMR-based studies of protein dynamics and molecular simulations have a synergistic relationship. Molecular simulations, in combination with interpretative theoretical models, leverage the dynamical information obtained from NMR. They provide the concrete physical schema underlying the quantities measured by NMR, and help extend the range of applications beyond the strictly dynamic properties. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.038DOI Listing
October 2018

Preface.

Authors:
A Joshua Wand

Methods Enzymol 2019 ;614:xv-xvii

Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1016/S0076-6879(18)30547-0DOI Listing
January 2019

A Quick and Colorful Method to Measure Low-Level Contaminations of Paramagnetic Ni in Protein Samples Purified by Immobilized Metal Ion Affinity Chromatography.

Methods Enzymol 2019 27;614:87-106. Epub 2018 Sep 27.

Department of Biochemistry & Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, United States. Electronic address:

Isotopic labeling of recombinantly expressed proteins is generally required for investigation by modern nuclear magnetic resonance (NMR) methods. Purification strategies of the labeled proteins often include the use of a polyhistidine affinity tag (His-tag) and immobilized metal ion affinity chromatography (IMAC). Described herein are rapid and inexpensive qualitative and quantitative assays to determine the concentration of paramagnetic Ni in protein samples purified by IMAC. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.037DOI Listing
September 2018

Optimal Isotope Labeling of Aromatic Amino Acid Side Chains for NMR Studies of Protein Dynamics.

Authors:
Ulrich Weininger

Methods Enzymol 2019 20;614:67-86. Epub 2018 Sep 20.

Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, Halle, Germany. Electronic address:

Aromatic side chains in proteins are often directly evolved in stabilizing the hydrophobic core, protein binding, or enzymatic activity. They are also responsible for specific local dynamic processes, such as histidine tautomerization or ring flips. Despite their importance, they are often not targeted directly by NMR spectroscopy, because of spectroscopic complications and challenges. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.028DOI Listing
September 2018

Combining NMR Spectroscopy and Molecular Dynamic Simulations to Solve and Analyze the Structure of Protein-RNA Complexes.

Methods Enzymol 2019 10;614:393-422. Epub 2018 Dec 10.

Department of Biology, ETH Zürich, Institute of Molecular Biology and Biophysics, Zürich, Switzerland. Electronic address:

Understanding the RNA binding specificity of protein is of primary interest to decipher their function in the cell. Here, we review the methodology used to solve the structures of protein-RNA complexes using solution-state NMR spectroscopy: from sample preparation to structure calculation procedures. We also describe how molecular dynamics simulations can help providing additional information on the role of key amino acid side chains and of water molecules in protein-RNA recognition. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.002DOI Listing
December 2018

Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics.

Methods Enzymol 2019 18;614:37-65. Epub 2018 Dec 18.

Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, TX, United States; Molecular Biophysics Graduate Program, The University of Texas Southwestern Medical Center, Dallas, TX, United States. Electronic address:

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.030DOI Listing
December 2018

Combining Evolutionary Covariance and NMR Data for Protein Structure Determination.

Methods Enzymol 2019 23;614:363-392. Epub 2018 Dec 23.

Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway, NJ, United States; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, NJ, United States; Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway, NJ, United States. Electronic address:

Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupled with advances in methods for sequence covariance analysis can provide reliable residue-residue contact information for a protein from sequence data alone. These "evolutionary couplings (ECs)" can be combined with sparse NMR data to determine accurate 3D protein structures. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.11.004DOI Listing
December 2018
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CS-ROSETTA.

Methods Enzymol 2019 11;614:321-362. Epub 2018 Sep 11.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA, United States. Electronic address:

Chemical Shift-Rosetta (CS-Rosetta) is an automated method that employs NMR chemical shifts to model protein structures de novo. In this chapter, we introduce the terminology and central concepts of CS-Rosetta. We describe the architecture and functionality of automatic NOESY assignment (AutoNOE) and structure determination protocols (Abrelax and RASREC) within the CS-Rosetta framework. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.07.005DOI Listing
September 2018

Exploring Protein Conformational Landscapes Using High-Pressure NMR.

Methods Enzymol 2019 12;614:293-320. Epub 2018 Dec 12.

Centre de Biochimie Structural CNRS Université de Montpellier UMR, Montpellier, France.

Protein conformational landscapes define their functional properties as well as their proteostasis. Hence, detailed mapping of these landscapes is necessary to understand and modulate protein conformation. The combination of high pressure and NMR provides a particularly powerful approach to characterizing protein conformational transitions. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.07.006DOI Listing
December 2018
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Nonuniform Sampling for NMR Spectroscopy.

Methods Enzymol 2019 26;614:263-291. Epub 2018 Oct 26.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States. Electronic address:

Nonuniform sampling was first proposed more than 40 years ago as an alternate method for sampling two-dimensional NMR data was initially pursued by only a small number of scientists. However, it has been gradually adopted after it was shown that major gains in measuring time and spectrum resolution can be obtained. Furthermore, migration of NMR software to the Unix environment facilitated development of new processing tools, and there is now a selection of programs available that yield high-quality reconstructions of NUS data. Read More

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https://linkinghub.elsevier.com/retrieve/pii/S00766879183036
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http://dx.doi.org/10.1016/bs.mie.2018.09.009DOI Listing
October 2018
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The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding.

Methods Enzymol 2019 22;614:239-261. Epub 2018 Sep 22.

Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA, United States.

The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.021DOI Listing
September 2018
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Synthesis and NMR Characterization of the Prenylated Peptide, a-Factor.

Methods Enzymol 2019 22;614:207-238. Epub 2018 Dec 22.

University of Minnesota, Twin Cities, Minneapolis, MN, United States. Electronic address:

Protein and peptide prenylation is an essential biological process involved in many signal transduction pathways. Hence, it plays a critical role in establishing many major human ailments, including Alzheimer's disease, amyotrophic lateral sclerosis (ALS), malaria, and Ras-related cancers. Yeast mating pheromone a-factor is a small dodecameric peptide that undergoes prenylation and subsequent processing in a manner identical to larger proteins. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.09.025DOI Listing
December 2018
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Preparation of Phosphorylated Proteins for NMR Spectroscopy.

Methods Enzymol 2019 5;614:187-205. Epub 2018 Sep 5.

Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, United States. Electronic address:

Phosphorylation is a ubiquitous posttranslational modification that is essential for the regulation of many cellular processes. The human genome consists of more than 200,000 phosphorylation sites, whose phosphorylation is tightly controlled by ≥500 kinases and ~200 phosphatases. Given the large number of phosphorylation sites and the key role phosphorylation plays in regulating cellular processes, it is essential to characterize the impact of phosphorylation on substrate structure, dynamics, and function. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.07.004DOI Listing
September 2018

Synthetic Biology-Based Solution NMR Studies on Membrane Proteins in Lipid Environments.

Methods Enzymol 2019 21;614:143-185. Epub 2018 Dec 21.

Institute of Biophysical Chemistry, Centre for Biomolecular Magnetic Resonance, J.W. Goethe-University, Frankfurt am Main, Germany. Electronic address:

Although membrane proteins are in the focus of biochemical research for many decades the general knowledge of this important class is far behind soluble proteins. Despite several recent technical developments, the most challenging feature still is the generation of high-quality samples in environments suitable for the selected application. Reconstitution of membrane proteins into lipid bilayers will generate the most native-like environment and is therefore commonly desired. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.019DOI Listing
December 2018

Toolkit for NMR Studies of Methyl-Labeled Proteins.

Methods Enzymol 2019 25;614:107-142. Epub 2018 Sep 25.

Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, United States. Electronic address:

Selective methyl labeling is an extremely powerful approach to study the structure, dynamics, and explore mechanistic insights of large biomolecules by solution NMR. Methyls are relatively insensitive to chemical exchange-induced depolarization and provide superior probes of supramolecular interactions and allostery in such systems. In this chapter, we describe our systematic approach and contributions in the areas of sample preparation, data collection, and data analysis that streamline the application of methyl labeling in solution NMR studies of large proteins. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.036DOI Listing
September 2018
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Selective Methyl Labeling of Proteins: Enabling Structural and Mechanistic Studies As Well As Drug Discovery Applications by Solution-State NMR.

Methods Enzymol 2019 21;614:1-36. Epub 2018 Dec 21.

Structural and Biophysical Chemistry, Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Emeryville, CA, United States; Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, Basel, Switzerland. Electronic address:

Escherichia coli expression protocols for selective labeling of methyl groups in proteins have been essential in expanding the size range of targets that can be studied by biomolecular NMR. Based on the initial work achieving selective labeling of isoleucine, leucine, and valine residues, additional methods were developed over the past years which enabled the individual and/or simultaneous combinatorial labeling of all methyl containing amino acids. Together with the introduction of new methyl-optimized NMR experiments, this now allows the detailed characterization of protein-ligand interactions as well as mechanistic and dynamic processes of protein-protein complexes up to 1MDa in size. Read More

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http://dx.doi.org/10.1016/bs.mie.2018.08.035DOI Listing
December 2018
1 Read