The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4... more The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4), a protein of-15 kDa which plays a key role in the regulation of B and T lymphocytes, has been determined using three-and four-dimensional heteronuclear N M R spectroscopy. The structure is based on a total of 2973 experimental N M R restraints, comprising 25 15 approximate interproton distance restraints, 102 distance restraints for 51 backbone hydrogen bonds, and 356 torsion angle restraints. A total of 30 structures was calculated by means of hybrid distance geometry-simulated annealing, and the atomic rms distribution about the mean coordinate positions for residues 8-129 is 0.44 f 0.03 A for the backbone atoms, 0.83 f 0.03 A for all atoms, and 0.51 f 0.04 A for all atoms excluding disordered side chains. The Nand C-terminal residues (1-7 and 130-133, respectively) appear to be disordered. The structure of IL-4 is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections. The linker elements between the helices are formed by either long loops, small helical turns, or short strands. The latter include a mini anti-parallel P-sheet. A best fit superposition of the N M R structure of IL-4 with the 2.25 A resolution crystal structure [Wlodawer, A., Pavlovsky, A., & Gutschina, A. (1992) FEBS Lett. 309, 59-64] yields a backbone atomic rms difference of 1.37 A which can be mainly attributed to tighter packing of the helices in the crystal structure. This is indicated by an approximately 20% reduction in the axial separation of three pairs of helices (CYA-CYC, CYA-CYD, and CYCCYD) in the crystal structure relative to the N M R structure and may reflect the greater flexibility of the molecule in solution which is reduced in the crystal due to intermolecular contacts. Comparison of the N M R structure of IL-4 with the X-ray structures ~ ~~ ~ ~~_ _ I Abbreviations: IL-4, human recombinant interleukin-4 (the numbering schemeused in the present articleincludes the four-residuesequence Glu-Ala-Glu-Ala at the N-terminus of the recombinant protein which is not part of the natural human IL-4; the natural sequence therefore starts at residue 5) ; NMR, nuclear magnetic resonance; Ig, immunoglobulin; HGH, human growth hormone; GM-CSF, granulocytemacrophage colony stimulating factor; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; ROE, rotating frame Overhauser effect; ROESY, rotating frame Overhauser enhancement spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; HMQC, heteronuclear multiple quantum coherence; CT, constant time; HCACO, CbH proton to a-carbon to carbonyl carbon correlation; HNHB, amide proton to nitrogen to CBH proton correlation; HN(CO)HB, amide proton to nitrogen to CBH proton (via carbonyl carbon); 3D, three-dimensional; 4D, four-dimensional; TPPI, timeproportional phase incrementation; SA, simulated annealing; rms, root mean square.
The functional evolution of proteins advances through gene duplication followed by functional dri... more The functional evolution of proteins advances through gene duplication followed by functional drift, whereas molecular evolution occurs through random mutational events. Over time, protein active-site structures or functional epitopes remain highly conserved, which enables relationships to be inferred between distant orthologs or paralogs. In this study, we present the first functional clustering and evolutionary analysis of the RCSB Protein Data Bank (RCSB PDB) based on similarities between active-site structures. All of the ligand-bound proteins within the RCSB PDB were scored using our Comparison of Protein Active-site Structures (CPASS) software and database (http://cpass.unl.edu/). Principal component analysis was then used to identify 4,431 representative structures to construct a phylogenetic tree based on the CPASS comparative scores (http://itol.embl.de/shared/jcatazaro). The resulting phylogenetic tree identified a sequential, stepwise evolution of protein active-sites and provides novel insights into the emergence of protein function or changes in substrate specificity based on subtle changes in geometry and amino acid composition.
Although the concepts of nonuniform sampling (NUS) and non-Fourier spectral reconstruction in mul... more Although the concepts of nonuniform sampling (NUS) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago (Bodenhausen and Ernst, 1981; Barna and Laue, 1987), it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection Published by Copernicus Publications on behalf of the Groupement AMPERE. 844 Y. Pustovalova et al.: NUScon of closely spaced peaks (i.e., "resolution") or peaks of weak intensity (i.e., "sensitivity"). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the "Nonuniform Sampling Contest" (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform.
Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The e... more Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effect...
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating pain disorder ... more Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating pain disorder of the bladder and urinary tract with poorly understood etiology. A definitive diagnosis of IC/BPS can be challenging because many symptoms are shared with other urological disorders. An analysis of urine presents an attractive and non-invasive resource for monitoring and diagnosing IC/BPS. The antiproliferative factor (APF) peptide has been previously identified in the urine of IC/BPS patients and is a proposed biomarker for the disorder. Nevertheless, other small urinary peptides have remained uninvestigated in IC/BPS primarily because protein biomarker discovery efforts employ protocols that remove small endogenous peptides. The purpose of this study is to investigate the profile of endogenous peptides in IC/BPS patient urine, with the goal of identifying putative peptide biomarkers. Here, a non-targeted peptidomics analysis of urine samples collected from IC/BPS patients were compare...
The steroidogenic acute regulatory-related lipid transfer (START) domain is found in both eukaryo... more The steroidogenic acute regulatory-related lipid transfer (START) domain is found in both eukaryotes and prokaryotes, with putative functions including signal transduction, transcriptional regulation, GTPase activation and thioester hydrolysis. Here we report the near complete 1H, 15N and 13C backbone and side chain NMR resonance assignments for the Bacillus subtilis START domain protein yndB
Clinical metabolomics is a rapidly expanding field focused on identifying molecular biomarkers to... more Clinical metabolomics is a rapidly expanding field focused on identifying molecular biomarkers to aid in the efficient diagnosis and treatment of human diseases. Variations in study design, metabolomics methodologies, and investigator protocols raise serious concerns about the accuracy and reproducibility of these potential biomarkers. The explosive growth of the field has led to the recent availability of numerous replicate clinical studies, which permits an evaluation of the consistency of biomarkers identified across multiple metabolomics projects. Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death and has the lowest five-year survival rate primarily due to the lack of an early diagnosis and the limited treatment options. Accordingly, PDAC has been a popular target of clinical metabolomics studies. We compiled 24 PDAC metabolomics studies from the scientific literature for a detailed meta-analysis. A consistent identification across these m...
Metabolomics investigates global metabolic alterations associated with chemical, biological, phys... more Metabolomics investigates global metabolic alterations associated with chemical, biological, physiological, or pathological processes. These metabolic changes are measured with various analytical platforms including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). While LC-MS methods are becoming increasingly popular in the field of metabolomics (accounting for more than 70% of published metabolomics studies to date), there are considerable benefits and advantages to NMR-based methods for metabolomic studies. In fact, according to PubMed, more than 926 papers on NMR-based metabolomics were published in 2021—the most ever published in a given year. This suggests that NMR-based metabolomics continues to grow and has plenty to offer to the scientific community. This perspective outlines the growing applications of NMR in metabolomics, highlights several recent advances in NMR technologies fo...
New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens an... more New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens and to expand the dwindling supply of effective antimicrobials. A common impediment to drug development is the lack of an easy approach to determine the in vivo mechanism of action and efficacy of novel drug leads. Towards this end, we describe an unbiased approach to predict in vivo mechanisms of action from NMR metabolomics data. Mycobacterium smegmatis, a nonpathogenic model organism for Mycobacterium tuberculosis, was treated with 12 known drugs and 3 chemical leads identified from a cell-based assay. NMR analysis of drug-induced changes to the M. smegmatis metabolome resulted in distinct clustering patterns correlating with in vivo drug activity. The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict in vivo activity
Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human path... more Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation
Structural genomics is providing a means to determine the molecular and cellular function for the... more Structural genomics is providing a means to determine the molecular and cellular function for the vast amount of proteins in the Human proteome that lack any explicit experimental information by characterizing the complete range of protein folds (Montelione, 2001). The Northeast Structural Genomics Consortium (NESG; http://www.nesg.org/) is a pilot project funded by the National Institutes of Health Protein Structure Initiative, focusing on proteins from eukaryotic model organisms including humans. The thermophillic archaea Archaeglobus fulgidis AF2095 protein is an example of a protein of unknown biological function targeted for structural analysis by NESG. AF2095 belongs to the Pfam family PF01981 – UPF0099, protein domain family of unknown function that has been found in yeast, archaebacteria and eubacteria. AF2095 has been assigned to NESG Cluster ID:17431, a set of fourteen proteins with high (\u3e~30%) sequence identity with human, Drosophila, Caenorhabditis elegans, Arabidops...
Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invas... more Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kin...
Parkinson\u27s disease (PD) is a neurodegenerative disease, which is characterized by progressive... more Parkinson\u27s disease (PD) is a neurodegenerative disease, which is characterized by progressive death of dopaminergic neurons in the substantia nigra pars compacta. Although mitochondrial dysfunction and oxidative stress are linked to PD pathogenesis, its etiology and pathology remain to be elucidated. Metabolomics investigates metabolite changes in biofluids, cell lysates, tissues and tumors in order to correlate these metabolomic changes to a disease state. Thus, the application of metabolomics to investigate PD provides a systematic approach to understand the pathology of PD, to identify disease biomarkers, and to complement genomics, transcriptomics and proteomics studies. This review will examine current research into PD mechanisms with a focus on mitochondrial dysfunction and oxidative stress. Neurotoxin-based PD animal models and the rationale for metabolomics studies in PD will also be discussed. The review will also explore the potential of NMR metabolomics to address imp...
Metabolomics is the comprehensive study of metabolism, the biochemical processes that sustain lif... more Metabolomics is the comprehensive study of metabolism, the biochemical processes that sustain life. By comparing metabolites between healthy and disease states, new insights into disease mechanisms can be uncovered. NMR is a powerful analytical method to detect and quantify metabolites. Standard one-dimensional (1D) 1H-NMR metabolite profiling is informative but challenged by significant chemical shift overlap. Multi-dimensional NMR can increase resolution, but the required long acquisition times lead to limited throughput. Non-uniform sampling (NUS) is a well-accepted mode of acquiring multi-dimensional NMR data, enabling either reduced acquisition times or increased sensitivity in equivalent time. Despite these advantages, the technique is not widely applied to metabolomics. In this study, we evaluated the utility of NUS 1H–13C heteronuclear single quantum coherence (HSQC) for semi-quantitative metabolomics. We demonstrated that NUS improved sensitivity compared to uniform samplin...
Proline utilization A (PutA) is a membrane associated multifunctional enzyme that catalyzes the o... more Proline utilization A (PutA) is a membrane associated multifunctional enzyme that catalyzes the oxidation of proline to glutamate in a two step process. In certain Gram-negative bacteria such as Pseudomonas putida, PutA also acts as an auto repressor in the cytoplasm when an insufficient concentration of proline is available. Here the N-terminal residues 1–45 of PutA from P. putida (PpPutA45), are shown to be responsible for DNA binding and dimerization. The solution structure of PpPutA45 was determined using NMR methods, where the protein is shown to be a symmetrical homodimer (12 kDa) consisting of two ribbon-helix-helix (RHH) structures. DNA sequence recognition by PpPutA45 was determined using DNA gel mobility shift assays and NMR chemical shift perturbations. PpPutA45 was shown to bind a 14 base-pair DNA oligomer (5′-GCGGTTGCACCTTT-3′). A model of the PpPutA45-DNA oligomer complex was generated using Haddock 2.1. The antiparallel β-sheet that results from PpPutA45 dimerization ...
Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCG... more Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ([MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine “rules” previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only...
An n ‒ Π* interaction between neighboring carbonyl groups has been postulated to stabilize protei... more An n ‒ Π* interaction between neighboring carbonyl groups has been postulated to stabilize protein structures. Such an interaction would affect the 13C chemical shielding of the carbonyl groups, whose paramagnetic component is dominated by n ‒ Π* and Π ‒ Π* excitations. Model compound calculations indicate that both the interaction energetics and the chemical shielding of the carbonyl group are instead dominated by a classical dipole-dipole interaction. A set of high resolution protein structures with associated carbonyl 13C chemical shift assignments verifies this correlation and provides no evidence for an inter-carbonyl n ‒ Π* interaction
The assignments of the 1H, 15N, 13CO, and 13C resonances of recombinant human basic fibroblast gr... more The assignments of the 1H, 15N, 13CO, and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation \u3e95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of Cα, Cβ, and Hα to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue cor-relation of Cα, Cβ, and Hα to the backbone amide 1H and 15N in the CBCANH and HNHA experi-ments. In addition, Cα and Cβ chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from th...
Structural genomics is providing a means to determ-ine the molecular and cellular function for th... more Structural genomics is providing a means to determ-ine the molecular and cellular function for the vast amount of proteins in the Human proteome that lack any explicit experimental information by characteriz-ing the complete range of protein folds (Montelione, 2001). The Northeast Structural Genomics Consor-tium (NESG; www.nesg.org) is a pilot project funded
The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4... more The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4), a protein of-15 kDa which plays a key role in the regulation of B and T lymphocytes, has been determined using three-and four-dimensional heteronuclear N M R spectroscopy. The structure is based on a total of 2973 experimental N M R restraints, comprising 25 15 approximate interproton distance restraints, 102 distance restraints for 51 backbone hydrogen bonds, and 356 torsion angle restraints. A total of 30 structures was calculated by means of hybrid distance geometry-simulated annealing, and the atomic rms distribution about the mean coordinate positions for residues 8-129 is 0.44 f 0.03 A for the backbone atoms, 0.83 f 0.03 A for all atoms, and 0.51 f 0.04 A for all atoms excluding disordered side chains. The Nand C-terminal residues (1-7 and 130-133, respectively) appear to be disordered. The structure of IL-4 is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections. The linker elements between the helices are formed by either long loops, small helical turns, or short strands. The latter include a mini anti-parallel P-sheet. A best fit superposition of the N M R structure of IL-4 with the 2.25 A resolution crystal structure [Wlodawer, A., Pavlovsky, A., & Gutschina, A. (1992) FEBS Lett. 309, 59-64] yields a backbone atomic rms difference of 1.37 A which can be mainly attributed to tighter packing of the helices in the crystal structure. This is indicated by an approximately 20% reduction in the axial separation of three pairs of helices (CYA-CYC, CYA-CYD, and CYCCYD) in the crystal structure relative to the N M R structure and may reflect the greater flexibility of the molecule in solution which is reduced in the crystal due to intermolecular contacts. Comparison of the N M R structure of IL-4 with the X-ray structures ~ ~~ ~ ~~_ _ I Abbreviations: IL-4, human recombinant interleukin-4 (the numbering schemeused in the present articleincludes the four-residuesequence Glu-Ala-Glu-Ala at the N-terminus of the recombinant protein which is not part of the natural human IL-4; the natural sequence therefore starts at residue 5) ; NMR, nuclear magnetic resonance; Ig, immunoglobulin; HGH, human growth hormone; GM-CSF, granulocytemacrophage colony stimulating factor; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; ROE, rotating frame Overhauser effect; ROESY, rotating frame Overhauser enhancement spectroscopy; HOHAHA, homonuclear Hartmann-Hahn spectroscopy; HMQC, heteronuclear multiple quantum coherence; CT, constant time; HCACO, CbH proton to a-carbon to carbonyl carbon correlation; HNHB, amide proton to nitrogen to CBH proton correlation; HN(CO)HB, amide proton to nitrogen to CBH proton (via carbonyl carbon); 3D, three-dimensional; 4D, four-dimensional; TPPI, timeproportional phase incrementation; SA, simulated annealing; rms, root mean square.
The functional evolution of proteins advances through gene duplication followed by functional dri... more The functional evolution of proteins advances through gene duplication followed by functional drift, whereas molecular evolution occurs through random mutational events. Over time, protein active-site structures or functional epitopes remain highly conserved, which enables relationships to be inferred between distant orthologs or paralogs. In this study, we present the first functional clustering and evolutionary analysis of the RCSB Protein Data Bank (RCSB PDB) based on similarities between active-site structures. All of the ligand-bound proteins within the RCSB PDB were scored using our Comparison of Protein Active-site Structures (CPASS) software and database (http://cpass.unl.edu/). Principal component analysis was then used to identify 4,431 representative structures to construct a phylogenetic tree based on the CPASS comparative scores (http://itol.embl.de/shared/jcatazaro). The resulting phylogenetic tree identified a sequential, stepwise evolution of protein active-sites and provides novel insights into the emergence of protein function or changes in substrate specificity based on subtle changes in geometry and amino acid composition.
Although the concepts of nonuniform sampling (NUS) and non-Fourier spectral reconstruction in mul... more Although the concepts of nonuniform sampling (NUS) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago (Bodenhausen and Ernst, 1981; Barna and Laue, 1987), it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection Published by Copernicus Publications on behalf of the Groupement AMPERE. 844 Y. Pustovalova et al.: NUScon of closely spaced peaks (i.e., "resolution") or peaks of weak intensity (i.e., "sensitivity"). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the "Nonuniform Sampling Contest" (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform.
Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The e... more Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effect...
Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating pain disorder ... more Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic and debilitating pain disorder of the bladder and urinary tract with poorly understood etiology. A definitive diagnosis of IC/BPS can be challenging because many symptoms are shared with other urological disorders. An analysis of urine presents an attractive and non-invasive resource for monitoring and diagnosing IC/BPS. The antiproliferative factor (APF) peptide has been previously identified in the urine of IC/BPS patients and is a proposed biomarker for the disorder. Nevertheless, other small urinary peptides have remained uninvestigated in IC/BPS primarily because protein biomarker discovery efforts employ protocols that remove small endogenous peptides. The purpose of this study is to investigate the profile of endogenous peptides in IC/BPS patient urine, with the goal of identifying putative peptide biomarkers. Here, a non-targeted peptidomics analysis of urine samples collected from IC/BPS patients were compare...
The steroidogenic acute regulatory-related lipid transfer (START) domain is found in both eukaryo... more The steroidogenic acute regulatory-related lipid transfer (START) domain is found in both eukaryotes and prokaryotes, with putative functions including signal transduction, transcriptional regulation, GTPase activation and thioester hydrolysis. Here we report the near complete 1H, 15N and 13C backbone and side chain NMR resonance assignments for the Bacillus subtilis START domain protein yndB
Clinical metabolomics is a rapidly expanding field focused on identifying molecular biomarkers to... more Clinical metabolomics is a rapidly expanding field focused on identifying molecular biomarkers to aid in the efficient diagnosis and treatment of human diseases. Variations in study design, metabolomics methodologies, and investigator protocols raise serious concerns about the accuracy and reproducibility of these potential biomarkers. The explosive growth of the field has led to the recent availability of numerous replicate clinical studies, which permits an evaluation of the consistency of biomarkers identified across multiple metabolomics projects. Pancreatic ductal adenocarcinoma (PDAC) is the third-leading cause of cancer-related death and has the lowest five-year survival rate primarily due to the lack of an early diagnosis and the limited treatment options. Accordingly, PDAC has been a popular target of clinical metabolomics studies. We compiled 24 PDAC metabolomics studies from the scientific literature for a detailed meta-analysis. A consistent identification across these m...
Metabolomics investigates global metabolic alterations associated with chemical, biological, phys... more Metabolomics investigates global metabolic alterations associated with chemical, biological, physiological, or pathological processes. These metabolic changes are measured with various analytical platforms including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). While LC-MS methods are becoming increasingly popular in the field of metabolomics (accounting for more than 70% of published metabolomics studies to date), there are considerable benefits and advantages to NMR-based methods for metabolomic studies. In fact, according to PubMed, more than 926 papers on NMR-based metabolomics were published in 2021—the most ever published in a given year. This suggests that NMR-based metabolomics continues to grow and has plenty to offer to the scientific community. This perspective outlines the growing applications of NMR in metabolomics, highlights several recent advances in NMR technologies fo...
New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens an... more New strategies are needed to circumvent increasing outbreaks of resistant strains of pathogens and to expand the dwindling supply of effective antimicrobials. A common impediment to drug development is the lack of an easy approach to determine the in vivo mechanism of action and efficacy of novel drug leads. Towards this end, we describe an unbiased approach to predict in vivo mechanisms of action from NMR metabolomics data. Mycobacterium smegmatis, a nonpathogenic model organism for Mycobacterium tuberculosis, was treated with 12 known drugs and 3 chemical leads identified from a cell-based assay. NMR analysis of drug-induced changes to the M. smegmatis metabolome resulted in distinct clustering patterns correlating with in vivo drug activity. The clustering of novel chemical leads relative to known drugs provides a mean to identify a protein target or predict in vivo activity
Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human path... more Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation
Structural genomics is providing a means to determine the molecular and cellular function for the... more Structural genomics is providing a means to determine the molecular and cellular function for the vast amount of proteins in the Human proteome that lack any explicit experimental information by characterizing the complete range of protein folds (Montelione, 2001). The Northeast Structural Genomics Consortium (NESG; http://www.nesg.org/) is a pilot project funded by the National Institutes of Health Protein Structure Initiative, focusing on proteins from eukaryotic model organisms including humans. The thermophillic archaea Archaeglobus fulgidis AF2095 protein is an example of a protein of unknown biological function targeted for structural analysis by NESG. AF2095 belongs to the Pfam family PF01981 – UPF0099, protein domain family of unknown function that has been found in yeast, archaebacteria and eubacteria. AF2095 has been assigned to NESG Cluster ID:17431, a set of fourteen proteins with high (\u3e~30%) sequence identity with human, Drosophila, Caenorhabditis elegans, Arabidops...
Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invas... more Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting that S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids, including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in the gudB gene, coding for glutamate dehydrogenase, which generates 2-oxoglutarate from glutamate, significantly reduced growth in CDM, suggesting that glutamate and those amino acids generating glutamate, particularly proline, serve as the major carbon source in this medium. Nuclear magnetic resonance (NMR) studies confirmed this supposition. Furthermore, a mutation in the ackA gene, coding for acetate kin...
Parkinson\u27s disease (PD) is a neurodegenerative disease, which is characterized by progressive... more Parkinson\u27s disease (PD) is a neurodegenerative disease, which is characterized by progressive death of dopaminergic neurons in the substantia nigra pars compacta. Although mitochondrial dysfunction and oxidative stress are linked to PD pathogenesis, its etiology and pathology remain to be elucidated. Metabolomics investigates metabolite changes in biofluids, cell lysates, tissues and tumors in order to correlate these metabolomic changes to a disease state. Thus, the application of metabolomics to investigate PD provides a systematic approach to understand the pathology of PD, to identify disease biomarkers, and to complement genomics, transcriptomics and proteomics studies. This review will examine current research into PD mechanisms with a focus on mitochondrial dysfunction and oxidative stress. Neurotoxin-based PD animal models and the rationale for metabolomics studies in PD will also be discussed. The review will also explore the potential of NMR metabolomics to address imp...
Metabolomics is the comprehensive study of metabolism, the biochemical processes that sustain lif... more Metabolomics is the comprehensive study of metabolism, the biochemical processes that sustain life. By comparing metabolites between healthy and disease states, new insights into disease mechanisms can be uncovered. NMR is a powerful analytical method to detect and quantify metabolites. Standard one-dimensional (1D) 1H-NMR metabolite profiling is informative but challenged by significant chemical shift overlap. Multi-dimensional NMR can increase resolution, but the required long acquisition times lead to limited throughput. Non-uniform sampling (NUS) is a well-accepted mode of acquiring multi-dimensional NMR data, enabling either reduced acquisition times or increased sensitivity in equivalent time. Despite these advantages, the technique is not widely applied to metabolomics. In this study, we evaluated the utility of NUS 1H–13C heteronuclear single quantum coherence (HSQC) for semi-quantitative metabolomics. We demonstrated that NUS improved sensitivity compared to uniform samplin...
Proline utilization A (PutA) is a membrane associated multifunctional enzyme that catalyzes the o... more Proline utilization A (PutA) is a membrane associated multifunctional enzyme that catalyzes the oxidation of proline to glutamate in a two step process. In certain Gram-negative bacteria such as Pseudomonas putida, PutA also acts as an auto repressor in the cytoplasm when an insufficient concentration of proline is available. Here the N-terminal residues 1–45 of PutA from P. putida (PpPutA45), are shown to be responsible for DNA binding and dimerization. The solution structure of PpPutA45 was determined using NMR methods, where the protein is shown to be a symmetrical homodimer (12 kDa) consisting of two ribbon-helix-helix (RHH) structures. DNA sequence recognition by PpPutA45 was determined using DNA gel mobility shift assays and NMR chemical shift perturbations. PpPutA45 was shown to bind a 14 base-pair DNA oligomer (5′-GCGGTTGCACCTTT-3′). A model of the PpPutA45-DNA oligomer complex was generated using Haddock 2.1. The antiparallel β-sheet that results from PpPutA45 dimerization ...
Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCG... more Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ([MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine “rules” previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only...
An n ‒ Π* interaction between neighboring carbonyl groups has been postulated to stabilize protei... more An n ‒ Π* interaction between neighboring carbonyl groups has been postulated to stabilize protein structures. Such an interaction would affect the 13C chemical shielding of the carbonyl groups, whose paramagnetic component is dominated by n ‒ Π* and Π ‒ Π* excitations. Model compound calculations indicate that both the interaction energetics and the chemical shielding of the carbonyl group are instead dominated by a classical dipole-dipole interaction. A set of high resolution protein structures with associated carbonyl 13C chemical shift assignments verifies this correlation and provides no evidence for an inter-carbonyl n ‒ Π* interaction
The assignments of the 1H, 15N, 13CO, and 13C resonances of recombinant human basic fibroblast gr... more The assignments of the 1H, 15N, 13CO, and 13C resonances of recombinant human basic fibroblast growth factor (FGF-2), a protein comprising 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation \u3e95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of Cα, Cβ, and Hα to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue cor-relation of Cα, Cβ, and Hα to the backbone amide 1H and 15N in the CBCANH and HNHA experi-ments. In addition, Cα and Cβ chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and Cα correlations from th...
Structural genomics is providing a means to determ-ine the molecular and cellular function for th... more Structural genomics is providing a means to determ-ine the molecular and cellular function for the vast amount of proteins in the Human proteome that lack any explicit experimental information by characteriz-ing the complete range of protein folds (Montelione, 2001). The Northeast Structural Genomics Consor-tium (NESG; www.nesg.org) is a pilot project funded
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Papers by Robert Powers