John Markley
University of Wisconsin-Madison, Biochemistry, Faculty Member
Research Interests: Biophysics, Chromatography, Biology, Crystallization, Capillary electrophoresis, and 15 moreBiological Sciences, Environmental Sciences, Automation, Animals, Cost effectiveness, Article, Affinity chromatography, Electrophoresis, Chemical Shift, Biomolecular, Amino Acid Sequence, Base Sequence, Affinity, Cell Growth, and culture medium
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<p>(A) (green) X-ray structure of the CobY<sup>G153D</sup>:GTP complex (PDB 3RSB) (87% structural overlap with rmsd 1.82 Å; fragment/topology score 0.95/0.94 with match size 113). (B) (gray) X-ray structure of... more
<p>(A) (green) X-ray structure of the CobY<sup>G153D</sup>:GTP complex (PDB 3RSB) (87% structural overlap with rmsd 1.82 Å; fragment/topology score 0.95/0.94 with match size 113). (B) (gray) X-ray structure of CMP:2-keto-3-deoxy-manno-octonic acid synthetase (PDB 1H7F).(75.5% structural overlap with rmsd 1.84 Å; fragment/topology score 0.93/1.0 with match size 148). (C) (gray) X-ray structure of α-D-glucose-1-phosphate cytidylyl transferase (PDB 1WVC) (75.5% structural overlap with rmsd 2.15 Å; fragment/topology score 0.86/1.0 with match size 148). (D) (gray) X-ray structure of acytidylyl transferase (PDB 2VSI) (74.5% structural overlap with rmsd 1.98 Å; fragment/topology score 0.91/1.0 with match size 146). (E) X-ray structure of 2-C-methyl-D-erythritol 4-phosphate cytidylyl transferase(PDB 1VPA) (73% structural overlap with rmsd 1.86 Å; fragment/topology score 0.91/1.0 with match size 143). (F) X-ray structure of CMP-acylneuraminate synthetase (PDB 1EYR) (70% structural overlap with rmsd 1.95 Å; fragment/topology score 0.88/1.0 with match size 137). PYMOL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141297#pone.0141297.ref020" target="_blank">20</a>] was used to generate the structures, and the CLICK [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141297#pone.0141297.ref033" target="_blank">33</a>] program was used to carry out the pairwise alignments.</p
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<p>The alignment, which was carried out using the PROMALS3D program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141297#pone.0141297.ref032" target="_blank">32</a>], shows... more
<p>The alignment, which was carried out using the PROMALS3D program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141297#pone.0141297.ref032" target="_blank">32</a>], shows substantial sequence alignment of secondary structural elements derived from the 3D structures: (blue) α-helix; (red) β-strand.</p
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Research Interests: Chemistry, Structure, Medicine, Biological Sciences, Cell, and 3 moreCHEMICAL SCIENCES, Cysteine, and Frataxin
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We have developed technology for producing accurate spectral fingerprints of small molecules through modeling of NMR spin system matrices to encapsulate their chemical shifts and scalar couplings. We describe here how libraries of these... more
We have developed technology for producing accurate spectral fingerprints of small molecules through modeling of NMR spin system matrices to encapsulate their chemical shifts and scalar couplings. We describe here how libraries of these spin systems utilizing unique and reproducible atom numbering can be used to improve NMR-based ligand screening and metabolomics studies. We introduce new Web services that facilitate the analysis of NMR spectra of mixtures of small molecules to yield their identification and quantification. The library of parametrized compounds has been expanded to cover simulations of H NMR spectra at a variety of magnetic fields of more than 1100 compounds, included are many common metabolites and a library of drug-like molecular fragments used in ligand screening. The compound library and related Web services are freely available from http://gissmo.nmrfam.wisc.edu/ .
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Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of... more
Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of internuclear vectors. We adapted a force field structure calculation methodology to allow free sampling of both R and S configurations of the stereocenters of interest. The algorithm uses a floating alignment tensor in a simulated annealing protocol to identify the conformations and configurations that best fit experimental RDC and distance restraints (from NOE and J-coupling data). A unique configuration (for rigid molecules) or a very small number of configurations (for less rigid molecules) of the structural models having the lowest chiral angle energies and reasonable magnitudes of the alignment tensor are provided as the best predictions of the unknown configuration. For highly flexible molecules, the progressive locking of their stereocenters into t...
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A polyether antibiotic, ecteinamycin (1) was isolated from a marine Actinomadura sp., cultivated from the ascidian Ectein-ascidia turbinata. 13C-enrichment, high resolution NMR spectroscopy and molecular modeling enabled elucidation of... more
A polyether antibiotic, ecteinamycin (1) was isolated from a marine Actinomadura sp., cultivated from the ascidian Ectein-ascidia turbinata. 13C-enrichment, high resolution NMR spectroscopy and molecular modeling enabled elucidation of the structure of 1 which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ec-teinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/μL), as well as other toxigenic and non-toxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On...
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Ferredoxins play an important role as an electron donor in iron-sulfur (Fe-S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human... more
Ferredoxins play an important role as an electron donor in iron-sulfur (Fe-S) cluster biosynthesis. Two ferredoxins, human mitochondrial ferredoxin 1 (FDX1) and human mitochondrial ferredoxin 2 (FDX2), are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron-sulfur cluster biogenesis. We report here biophysical studies of the interaction of these two ferredoxins with other proteins involved in mitochondrial iron-sulfur cluster assembly. Results from nuclear magnetic resonance spectroscopy show that both FDX1 and FDX2 (in both their reduced and oxidized states) interact with the protein complex responsible for cluster assembly, which contains cysteine desulfurase (NFS1), ISD11 (also known as LYRM4), and acyl carrier protein (Acp). In all cases, ferredoxin residues close to the Fe-S cluster are involved in the interaction with this complex. Isothermal titration calorimetry results showed that FDX2...
Research Interests: Biochemistry, Evolutionary Biology, Biophysics, Chemistry, Immunology, and 15 moreBiotechnology, Biology, Cell Biology, Humans, Escherichia coli, Animals, Biogenesis, Electrons, Cysteine, Amino Acid Sequence, American Chemical Society, Biochemistry and cell biology, Gene Expression Regulation, Acyl carrier protein, and Ferredoxin
Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried... more
Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (K...
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NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps... more
NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that makes biomolecular NMR spectroscopy much more accessible by integrating tools, databases, web services, and video tutorials that can be launched by simple installation of NMRFAM software packages or using a cross-platform virtual machine that can be run on any standard laptop or desktop computer. The software package can be downloaded freely from the NMRFAM software download page ( http://pine.nmrfam.wisc.edu/download_packages.html ), and detailed instructions are available from the Integrative NMR Video Tutorial page ( http://pine.nmrfam.wisc.edu/integrative.html ).
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NMR ligand affinity screening is a powerful technique that is routinely used in drug discovery or functional genomics to directly detect protein-ligand binding events. Binding events can be identified by monitoring differences in the... more
NMR ligand affinity screening is a powerful technique that is routinely used in drug discovery or functional genomics to directly detect protein-ligand binding events. Binding events can be identified by monitoring differences in the one-dimensional (1)H NMR spectrum of a compound with and without protein. Although a single NMR spectrum can be collected within a short period (2-10 min per sample), one-by-one screening of a protein against a library of hundreds or thousands of compounds requires a large amount of spectrometer time and a large quantity of protein. To improve the efficiency of these screens in both time and material, compounds are usually evaluated in mixtures ranging in size from 3 to 20 compounds. Ideally, the NMR signals from individual compounds in the mixture should not overlap so that spectral changes can be associated with a particular compound. We have developed a software tool, NMRmix, to assist in creating ideal mixtures from a large panel of compounds with k...
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IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two... more
IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural informatio...
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MolProbity is a powerful software program for validating structures of proteins and nucleic acids. Although MolProbity includes scripts for batch analysis of structures, because these scripts analyze structures one at a time, they are not... more
MolProbity is a powerful software program for validating structures of proteins and nucleic acids. Although MolProbity includes scripts for batch analysis of structures, because these scripts analyze structures one at a time, they are not well suited for the validation of a large dataset of structures. We have created a version of MolProbity (MolProbity-HTC) that circumvents these limitations and takes advantage of a high-throughput computing cluster by using the HTCondor software. MolProbity-HTC enables the longitudinal analysis of large sets of structures, such as those deposited in the PDB or generated through theoretical computation-tasks that would have been extremely time-consuming using previous versions of MolProbity. We have used MolProbity-HTC to validate the entire PDB, and have developed a new visual chart for the BioMagResBank website that enables users to easily ascertain the quality of each model in an NMR ensemble and to compare the quality of those models to the res...
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ABSTRACT We developed a new relational database management system called PACSY (Protein structure And Chemical Shift NMR spectroscopY) by integrating information from the Protein Data Bank (PDB), the Biological Magnetic Resonance Data... more
ABSTRACT We developed a new relational database management system called PACSY (Protein structure And Chemical Shift NMR spectroscopY) by integrating information from the Protein Data Bank (PDB), the Biological Magnetic Resonance Data Bank (BMRB), and the Structural Classification of Proteins (SCOP) database. PACSY offers valuable information for structural investigations such as three-dimensional coordinates and chemical shifts of atoms along with derived information such as torsion angles, solvent accessible surface areas, and hydrophobicity scales. PACSY consists of six relational table types linked to one another for coherence by key identification numbers. The database can be installed on an RDBMS server such as MySQL and PostgreSQL for advanced search functions by supporting database queries. PACSY enables users to search for combinations of information from different database sources in support of their research. PACSY along with two associated software packages, PACSY Maker for database creation and PACSY Analyzer for database analysis, are available from http://pacsy.nmrfam.wisc.edu.
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The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion... more
The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.
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How Sweet It Is: Detailed Molecular and Functional ... Studies of Brazzein, a Sweet Protein and Its Analogs ... Fariba Assadi-Porter1,2, Marco Tonelli2, James T. Radek3, Claudia C. Cornilescu4, and John L. Markley1,2 ... 1Department of... more
How Sweet It Is: Detailed Molecular and Functional ... Studies of Brazzein, a Sweet Protein and Its Analogs ... Fariba Assadi-Porter1,2, Marco Tonelli2, James T. Radek3, Claudia C. Cornilescu4, and John L. Markley1,2 ... 1Department of Biochemistry, 2National ...
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Research Interests: Biochemistry, Biology, Medicine, Carbon Isotopes, Escherichia coli, and 15 moreNmr, Affinity chromatography, Nmr Structure, Nitrogen Isotopes, Phosphodiesterase Inhibitors, Glycerol, HSQC, Atomic Structure, Large Scale, Amino Acid Sequence, Isotope Labeling, Full Length Movies, Biochemistry and cell biology, Molecular Sequence Data, and GTP
Research Interests: Computer Science, Algorithms, Computational Complexity, Protein Folding, Protein Structure Prediction, and 15 moreNuclear Magnetic Resonance, NMR Spectroscopy, Magnetic Resonance Spectroscopy, Medicine, Biological Sciences, Mathematical Sciences, Research article, Proteins, Protein Secondary Structure, Chemical Shift, Quasi Stationary States, Protein Binding, Isotope Labeling, Interaction Network, and binding sites
Research Interests: Chemistry, Biomolecular NMR, Computational Biology, Medicine, Biological Sciences, and 15 moreCarbon Isotopes, Physical sciences, Data Collection, Nitrogen Isotopes, Anisotropic media, CHEMICAL SCIENCES, High Resolution, Lower Bound, Molecular Conformation, Protein Conformation, Biomolecular, Multi Dimensional, Dipolar Coupling, Internet, and Biological System
Research Interests: Chemistry, Biomolecular NMR, Medicine, Biological Sciences, Dynamic Analysis, and 14 moreCarbon Isotopes, Scrambling, Physical sciences, Proteins, Nmr Structure, Nitrogen Isotopes, Enzyme, CHEMICAL SCIENCES, Isotope Dilution, Reproducibility of Results, Amino Acid Profile, Biomolecular, Molecular weight, and Amino Acid Sequence
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Research Interests: Biochemistry, Chemistry, Crystallography, Medicine, Molecular chaperones, and 13 moreEscherichia coli, Iron, Sulfur, Heat Shock Proteins, Amino Acid Substitution Rates, Protein Quaternary Structure, Protein Binding, Solution Structure, Biochemistry and cell biology, Nuclear Magnetic Resonance Spectroscopy, Linker, binding sites, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Chemistry, Nuclear Magnetic Resonance, Magnetic Resonance Spectroscopy, Medicine, and 11 moreEscherichia coli, Deoxyribonucleic Acid, Temperature, Spectrum, Proton, Base Sequence, Nucleic Acid Conformation, Oligonucleotides, Biochemistry and cell biology, Lambda, and Medical biochemistry and metabolomics
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Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small... more
Metabolites present in liver provide important clues regarding the physiological state of an organism. The aim of this work was to evaluate a protocol for high-throughput NMR-based analysis of polar and non-polar metabolites from a small quantity of liver tissue. We extracted the tissue with a methanol/chloroform/water mixture and isolated the polar metabolites from the methanol/water layer and the non-polar metabolites from the chloroform layer. Following drying, we re-solubilized the fractions for analysis with a 600 MHz NMR spectrometer equipped with a 1.7 mm cryogenic probe. In order to evaluate the feasibility of this protocol for metabolomics studies, we analyzed the metabolic profile of livers from house sparrow (Passer domesticus) nestlings raised on two different diets: livers from 10 nestlings raised on a high protein diet (HP) for 4 d and livers from 12 nestlings raised on the HP diet for 3 d and then switched to a high carbohydrate diet (HC) for 1 d. The protocol enabled...
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The editors of this special volume suggested this topic, presumably because of the perspective lent by our combined >90-year association with biomolecular NMR. What follows is our personal experience with the evolution of the field,... more
The editors of this special volume suggested this topic, presumably because of the perspective lent by our combined >90-year association with biomolecular NMR. What follows is our personal experience with the evolution of the field, which we hope will illustrate the trajectory of change over the years. As for the future, one can confidently predict that it will involve unexpected advances. Our narrative is colored by our experience in using the NMR Facility for Biomedical Studies at Carnegie-Mellon University (Pittsburgh) and in developing similar facilities at Purdue (1977-1984) and the University of Wisconsin-Madison (1984-). We have enjoyed developing NMR technology and making it available to collaborators and users of these facilities. Our group's association with the Biological Magnetic Resonance data Bank (BMRB) and with the Worldwide Protein Data Bank (wwPDB) has also been rewarding. Of course, many groups contributed to the early growth and development of biomolecular...
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Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N- and C-terminal domains of human NFU1... more
Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N- and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SAXS) data to derive structural models for full-length monomeric apo-NFU1, dimeric apo-NFU1 (an artifact of intermolecular disulfide bond formation), and holo-NFUI (the [4Fe-4S] cluster-containing form of the protein). Apo-NFU1 contains two cysteine residues in its C-terminal domain, and two apo-NFU1 subunits coordinate one [4Fe-4S] cluster to form a cluster-linked dimer. Holo-NFU1 consists of a complex of three of these dimers as shown by molecular weight estimates from SAXS and size-exclusion chromatography. The SAXS-derived structural model indicates that one N-terminal region from each of the three dimers forms a tripartite interface. Th...
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The two leading analytical approaches to metabolomics are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Although currently overshadowed by MS in terms of numbers of compounds resolved, NMR spectroscopy offers... more
The two leading analytical approaches to metabolomics are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Although currently overshadowed by MS in terms of numbers of compounds resolved, NMR spectroscopy offers advantages both on its own and coupled with MS. NMR data are highly reproducible and quantitative over a wide dynamic range and are unmatched for determining structures of unknowns. NMR is adept at tracing metabolic pathways and fluxes using isotope labels. Moreover, NMR is non-destructive and can be utilized in vivo. NMR results have a proven track record of translating in vitro findings to in vivo clinical applications.
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ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) represents a groundbreaking prototype for automated protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. With... more
ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) represents a groundbreaking prototype for automated protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. With a [(13)C,(15)N]-labeled protein sample loaded into the NMR spectrometer, ADAPT-NMR delivers complete backbone resonance assignments and secondary structure in an optimal fashion without human intervention. ADAPT-NMR achieves this by implementing a strategy in which the goal of optimal assignment in each step determines the subsequent step by analyzing the current sum of available data. ADAPT-NMR is the first iterative and fully automated approach designed specifically for the optimal assignment of proteins with fast data collection as a byproduct of this goal. ADAPT-NMR evaluates the current spectral information, and uses a goal-directed objective function to select the optimal next data collection step(s) and then directs the NMR spectrometer to co...
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Research Interests: Bioinformatics, Algorithms, Computer Graphics, Computational Biology, NMR Spectroscopy, and 14 moreProbability, Biological Sciences, Software, Assignment, Automation, Mathematical Sciences, Method, San Francisco, Proteins, Computer User Interface Design, Spectrum, Source Code, Chemical Shift, and Graphical User Interface
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Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been... more
Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we review our recently developed methods that greatly simplify PPI studies, which minimize the number of samples required to fully characterize residues involved in the protein-protein binding interface. This original strategy combines asymmetric labeling of two binding partners and the carbonyl-carbon label selective (CCLS) pulse sequence element implemented into the heteronuclear single quantum correlation (¹H-N HSQC) spectra. The CCLS scheme removes signals of the J-coupled N⁻C resonances and records simultaneously two individual amide fingerprints for each binding partner. We show the application to the measurements of chemical shift corr...
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Synaptosomes are isolated nerve terminals that contain synaptic components, including neurotransmitters, metabolites, adhesion/fusion proteins, and nerve terminal receptors. The essential role of synaptosomes in neurotransmission has... more
Synaptosomes are isolated nerve terminals that contain synaptic components, including neurotransmitters, metabolites, adhesion/fusion proteins, and nerve terminal receptors. The essential role of synaptosomes in neurotransmission has stimulated keen interest in understanding both their proteomic and metabolic composition. Mass spectrometric (MS) quantification of synaptosomes has illuminated their proteomic composition, but the determination of the metabolic composition by MS has been met with limited success. In this study, we report a proof-of-concept application of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy for analyzing the metabolic composition of synaptosomes. We utilize this approach to compare the metabolic composition synaptosomes from a wild-type rat with that from a newly generated genetic rat model (Disc1 svΔ2), which qualitatively recapitulates clinically observed early DISC1 truncations associated with schizophrenia. This study demonstrates ...
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Iron-sulfur (Fe-S) clusters, the ubiquitous protein cofactors found in all kingdoms of life, perform a myriad of functions including nitrogen fixation, ribosome assembly, DNA repair, mitochondrial respiration, and metabolite catabolism.... more
Iron-sulfur (Fe-S) clusters, the ubiquitous protein cofactors found in all kingdoms of life, perform a myriad of functions including nitrogen fixation, ribosome assembly, DNA repair, mitochondrial respiration, and metabolite catabolism. The biogenesis of Fe-S clusters is a multi-step process that involves the participation of many protein partners. Recent biophysical studies, involving X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and small angle X-ray scattering (SAXS), have greatly improved our understanding of these steps. In this review, after describing the biological importance of iron sulfur proteins, we focus on the contributions of NMR spectroscopy has made to our understanding of the structures, dynamics, and interactions of proteins involved in the biosynthesis of Fe-S cluster proteins.
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The constant-relaxation-period (CREPE) method for spin-lattice (T1) relaxation measurement presented here offers a significant improvement over other methods currently in use. The CREPE approach involves the measurement of both the... more
The constant-relaxation-period (CREPE) method for spin-lattice (T1) relaxation measurement presented here offers a significant improvement over other methods currently in use. The CREPE approach involves the measurement of both the initial and the final magnetizations flanking a constant relaxation period; the relaxation rates can be extracted from the data by linear regression without the necessity of additional parameters to correct for off-resonance effects or errors in pulse-width settings. Computer simulations used to compare the accuracy of T1 measurements made by the new method with those by the fast inversion-recovery (FIR) approach (generally considered to be the best general method for T1 measurements) showed that the CREPE method provides about a twofold reduction in errors over a wide range of signal-to-noise in the input data. Experimental data collected by the two approaches showed that the CREPE method provides the same precision in T1 values as the FIR method with one-fourth the data-collection time.