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Pubmed Article
Solution NMR structure and histone binding of the PHD domain of human MLL5.
PLoS ONE
PUBLISHED: 01-01-2013
Mixed Lineage Leukemia 5 (MLL5) is a histone methyltransferase that plays a key role in hematopoiesis, spermatogenesis and cell cycle progression. In addition to its catalytic domain, MLL5 contains a PHD finger domain, a protein module that is often involved in binding to the N-terminus of histone H3. Here we report the NMR solution structure of the MLL5 PHD domain showing a variant of the canonical PHD fold that combines conserved H3 binding features from several classes of other PHD domains (including an aromatic cage) along with a novel C-terminal ?-helix, not previously seen. We further demonstrate that the PHD domain binds with similar affinity to histone H3 tail peptides di- and tri-methylated at lysine 4 (H3K4me2 and H3K4me3), the former being the putative product of the MLL5 catalytic reaction. This work establishes the PHD domain of MLL5 as a bone fide reader domain of H3K4 methyl marks suggesting that it may guide the spreading or further methylation of this site on chromatin.
Authors: Michael L. Beshiri, Abul Islam, Dannielle C. DeWaal, William F. Richter, Jennifer Love, Nuria Lopez-Bigas, Elizaveta V. Benevolenskaya.
Published: 07-07-2010
ABSTRACT
Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.
19 Related JoVE Articles!
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The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Authors: Monica Soldi, Tiziana Bonaldi.
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
51220
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Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Authors: Johan Nilvebrant, Tove Alm, Sophia Hober.
Institutions: Royal Institute of Technology.
Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2. Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 . The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix. This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.
Molecular Biology, Issue 59, Affinity chromatography, albumin-binding domain, human serum albumin, Z-domain
3370
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Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation
Authors: Raja S. Vasireddy, Michelle M. Tang, Li-Jeen Mah, George T. Georgiadis, Assam El-Osta, Tom C. Karagiannis.
Institutions: The Alfred Medical Research and Education Precinct, The Alfred Medical Research and Education Precinct, University of Melbourne.
An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4. Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.
Cellular Biology, Issue 42, H2AX, radiation, euchromatin, heterochromatin, immunofluorescence, 3D-modeling
2203
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Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
Authors: Catherine Joce, Alyssa Wiener, Hang Yin.
Institutions: University of Colorado at Boulder.
The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven. In many cases membrane-spanning proteins have evolved highly sophisticated mechanisms of action.1-3 One way in which membrane proteins can modulate their structures and functions is by direct and specific contact of hydrophobic helices, forming structured transmembrane oligomers.4,5 Much recent work has focused on the distribution of amino acids preferentially found in the membrane environment in comparison to aqueous solution and the different intermolecular forces that drive protein association.6,7 Nevertheless, studies of molecular recognition at the transmembrane domain of proteins still lags behind those of water-soluble regions. A major hurdle remains: despite the remarkable specificity and affinity that transmembrane oligomerization can achieve,8 direct measurement of their association is challenging. Traditional methodologies applied to the study of integral membrane protein function can be hampered by the inherent insolubility of the sequences under examination. Biophysical insights gained from studying synthetic peptides representing transmembrane domains can provide useful structural insight. However, the biological relevance of the detergent micellar or liposome systems used in these studies to mimic cellular membranes is often questioned; do peptides adopt a native-like structure under these conditions and does their functional behaviour truly reflect the mode of action within a native membrane? In order to study the interactions of transmembrane sequences in natural phospholipid bilayers, the Langosch lab developed ToxR transcriptional reporter assays.9 The transmembrane domain of interest is expressed as a chimeric protein with maltose binding protein for location to the periplasm and ToxR to provide a report of the level of oligomerization (Figure 1). In the last decade, several other groups (e.g. Engelman, DeGrado, Shai) further optimized and applied this ToxR reporter assay.10-13 The various ToxR assays have become a gold standard to test protein-protein interactions in cell membranes. We herein demonstrate a typical experimental operation conducted in our laboratory that primarily follows protocols developed by Langosch. This generally applicable method is useful for the analysis of transmembrane domain self-association in E. coli, where β-galactosidase production is used to assess the TMD oligomerization propensity. Upon TMD-induced dimerization, ToxR binds to the ctx promoter causing up-regulation of the LacZ gene for β-galactosidase. A colorimetric readout is obtained by addition of ONPG to lyzed cells. Hydrolytic cleavage of ONPG by β-galactosidase results in the production of the light absorbing species o-nitrophenolate (ONP) (Figure 2).
Cellular Biology, Issue 51, Transmembrane domain, oligomerization, transcriptional reporter, ToxR, latent membrane protein-1
2721
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Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR
Authors: Michal S. Shoshan, Edit Y. Tshuva, Deborah E. Shalev.
Institutions: The Hebrew University of Jerusalem, The Hebrew University of Jerusalem.
Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy. NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.
Chemistry, Issue 82, solution structure determination, NMR, peptide models, copper-binding proteins, copper complexes
50747
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A Protocol for Computer-Based Protein Structure and Function Prediction
Authors: Ambrish Roy, Dong Xu, Jonathan Poisson, Yang Zhang.
Institutions: University of Michigan , University of Kansas.
Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently >20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.
Biochemistry, Issue 57, On-line server, I-TASSER, protein structure prediction, function prediction
3259
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Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies
Authors: Pooja Sharma, Mariam Kaywan-Lutfi, Logesvaran Krshnan, Eamon F. X. Byrne, Melissa Joy Call, Matthew Edwin Call.
Institutions: The Walter and Eliza Hall Institute of Medical Research, The University of Melbourne.
Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.
Biochemistry, Issue 73, Structural Biology, Chemistry, Chemical Engineering, Biophysics, Genetics, Molecular Biology, Membrane Proteins, Proteins, Molecular Structure, transmembrane domain, peptide chemistry, membrane protein structure, immune receptors, reversed-phase HPLC, HPLC, peptides, lipids, protein, cloning, TFA Elution, CNBr Digestion, NMR, expression, cell culture
50141
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Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins
Authors: Kevin J. Roberson, Pamlea N. Brady, Michelle M. Sweeney, Megan A. Macnaughtan.
Institutions: Louisiana State University.
Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein's primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein's N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.
Chemistry, Issue 82, Boranes, Formaldehyde, Dimethylamines, Tandem Mass Spectrometry, nuclear magnetic resonance, MALDI-TOF, Reductive methylation, lysozyme, dimethyllysine, mass spectrometry, NMR
50875
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Detection of Histone Modifications in Plant Leaves
Authors: Michal Jaskiewicz, Christoph Peterhansel, Uwe Conrath.
Institutions: RWTH Aachen University, RWTH Aachen University, Leibniz University.
Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.
Molecular Biology, Issue 55, chromatin, chromatin immunoprecipitation, ChIP, histone modifications, PCR, plant molecular biology, plant promoter control, gene regulation
3096
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Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells
Authors: Geoffrey Berguet, Jan Hendrickx, Celine Sabatel, Miklos Laczik, Sharon Squazzo, Ignacio Mazon Pelaez, Rini Saxena, Helene Pendeville, Dominique Poncelet.
Institutions: Diagenode S.A., Diagenode Inc..
Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.
Molecular Biology, Issue 94, Automation, chromatin immunoprecipitation, low DNA amounts, histone antibodies, sequencing, library preparation
52150
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Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity
Authors: Lauren P. Blair, Nathan L. Avaritt, Alan J. Tackett.
Institutions: University of Arkansas for Medical Sciences .
Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity. Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.
Molecular Biology, Issue 61, LSD1, lysine demethylase, mass spectrometry, reductive methylation, demethylase quantification
3604
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Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays
Authors: Srikanth Kudithipudi, Denis Kusevic, Sara Weirich, Albert Jeltsch.
Institutions: Stuttgart University.
Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.
Biochemistry, Issue 93, Peptide arrays, solid phase peptide synthesis, SPOT synthesis, protein lysine methyltransferases, substrate specificity profile analysis, lysine methylation
52203
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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
Authors: Gisela Maria Hanz, Britta Jung, Anna Giesbertz, Matyas Juhasz, Elmar Weinhold.
Institutions: RWTH Aachen University.
S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5’-ATCGAT-3’ sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.
Biochemistry, Issue 93, S-adenosyl-l-methionine, AdoMet, SAM, aziridine cofactor, double activated cofactor, methyltransferase, DNA methylation, protein methylation, biotin labeling, fluorescence labeling, SMILing, mTAG
52014
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Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Authors: James Smadbeck, Meghan B. Peterson, George A. Khoury, Martin S. Taylor, Christodoulos A. Floudas.
Institutions: Princeton University.
The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity. To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
50476
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Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA
Authors: Ryan A. Rogge, Anna A. Kalashnikova, Uma M. Muthurajan, Mary E. Porter-Goff, Karolin Luger, Jeffrey C. Hansen.
Institutions: Colorado State University .
Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.
Cellular Biology, Issue 79, Chromosome Structures, Chromatin, Nucleosomes, Histones, Microscopy, Atomic Force (AFM), Biochemistry, Chromatin, Nucleosome, Nucleosomal Array, Histone, Analytical Ultracentrifugation, Sedimentation Velocity
50354
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Biochemical Assays for Analyzing Activities of ATP-dependent Chromatin Remodeling Enzymes
Authors: Lu Chen, Soon-Keat Ooi, Joan W. Conaway, Ronald C. Conaway.
Institutions: Stowers Institute for Medical Research, Kansas University Medical Center.
Members of the SNF2 family of ATPases often function as components of multi-subunit chromatin remodeling complexes that regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Biochemically dissecting the contributions of individual subunits of such complexes to the multi-step ATP-dependent chromatin remodeling reaction requires the use of assays that monitor the production of reaction products and measure the formation of reaction intermediates. This JOVE protocol describes assays that allow one to measure the biochemical activities of chromatin remodeling complexes or subcomplexes containing various combinations of subunits. Chromatin remodeling is measured using an ATP-dependent nucleosome sliding assay, which monitors the movement of a nucleosome on a DNA molecule using an electrophoretic mobility shift assay (EMSA)-based method. Nucleosome binding activity is measured by monitoring the formation of remodeling complex-bound mononucleosomes using a similar EMSA-based method, and DNA- or nucleosome-dependent ATPase activity is assayed using thin layer chromatography (TLC) to measure the rate of conversion of ATP to ADP and phosphate in the presence of either DNA or nucleosomes. Using these assays, one can examine the functions of subunits of a chromatin remodeling complex by comparing the activities of the complete complex to those lacking one or more subunits. The human INO80 chromatin remodeling complex is used as an example; however, the methods described here can be adapted to the study of other chromatin remodeling complexes.
Biochemistry, Issue 92, chromatin remodeling, INO80, SNF2 family ATPase, biochemical assays, ATPase, nucleosome remodeling, nucleosome binding
51721
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Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Authors: Kristen K. McCampbell, Kristin N. Springer, Rebecca A. Wingert.
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90, zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
51644
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Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
Authors: Matthew Rames, Yadong Yu, Gang Ren.
Institutions: The Molecular Foundry.
Structural determination of proteins is rather challenging for proteins with molecular masses between 40 - 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 - 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3 . Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high‐resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high‐throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.
Environmental Sciences, Issue 90, small and asymmetric protein structure, electron microscopy, optimized negative staining
51087
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Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin
Authors: Jyoti Srivastava, Diane Barber.
Institutions: University of California, San Francisco - UCSF.
The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.
Biochemistry, Issue 13, F-actin, protein, in vitro binding, ultracentrifugation
690
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