[58] C. F. DeLisle, H. B. Mendis, and J. L Lorieau. Super resolution noesy spectra of proteins. Journal of Biomolecular NMR, (In Press), 2019.
No abstract.
[57] CongBao Kang. Applications of in-cell NMR in structural biology and drug discovery. International Journal of Molecular Sciences, 20(1):139, Jan 2019. [ DOI | www: ]
In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein--protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.
[56] Marion Pupier, Jean-Marc Nuzillard, Julien Wist, Nils E. Schlörer, Stefan Kuhn, Mate Erdelyi, Christoph Steinbeck, Antony J. Williams, Craig Butts, Tim D.W. Claridge, Bozhana Mikhova, Wolfgang Robien, Hesam Dashti, Hesam, Hamid R. Eghbalnia, Christophe Farès, Christian Adam, Pavel Kessler, Fabrice Moriaud, Mikhail Elyashberg, Dimitris Argyropoulos, Manuel Pérez, Patrick Giraudeau, Roberto R. Gil, Paul Trevorrow, and Damien Jeannerat. NMReDATA, a standard to report the NMR assignment and parameters of organic compounds. Magnetic Resonance in Chemistry, 56(8):703--715, May 2018. [ DOI ]
Even though NMR has found countless applications in the field of small molecule characterization, there is no standard file format available for the NMR data relevant to structure characterization of small molecules. A new format is therefore introduced to associate the NMR parameters extracted from 1D and 2D spectra of organic compounds to the proposed chemical structure. These NMR parameters, which we shall call NMReDATA (for nuclear magnetic resonance extracted data), include chemical shift values, signal integrals, intensities, multiplicities, scalar coupling constants, lists of 2D correlations, relaxation times, and diffusion rates. The file format is an extension of the existing Structure Data Format, which is compatible with the commonly used MOL format. The association of an NMReDATA file with the raw and spectral data from which it originates constitutes an NMR record. This format is easily readable by humans and computers and provides a simple and efficient way for disseminating results of structural chemistry investigations, allowing automatic verification of published results, and for assisting the constitution of highly needed open‐source structural databases.
[55] João M. C. Teixeira, Simon P. Skinner, Miguel Arbesú, Alexander L. Breeze, and Miquel Pons. Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data. Journal of Biomolecular NMR, 71(1):1--9, May 2018. [ DOI ]
We present Farseer-NMR (, a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution. These advances have created a growing need to analyse those systems' responses to multiple variables. The combined analysis of NMR peaklists from large and multivariable datasets has become a new bottleneck in the NMR analysis pipeline, whereby information-rich NMR-derived parameters have to be manually generated, which can be tedious, repetitive and prone to human error, or even unfeasible for very large datasets. There is a persistent gap in the development and distribution of software focused on peaklist treatment, analysis and representation, and specifically able to handle large multivariable datasets, which are becoming more commonplace. In this regard, Farseer-NMR aims to close this longstanding gap in the automated NMR user pipeline and, altogether, reduce the time burden of analysis of large sets of peaklists from days/weeks to seconds/minutes. We have implemented some of the most common, as well as new, routines for calculation of NMR parameters and several publication-quality plotting templates to improve NMR data representation. Farseer-NMR has been written entirely in Python and its modular code base enables facile extension.
[54] Guillaume Laurent and Christian Bonhomme. Non-uniform sampling applied to solid-state NMR. In RMN structurale dans le Bassin Parisien, Orléans, France, March 2018. [ http ]
No abstract.
[53] Matthew A Zambrello, Adam D Schuyler, Mark W Maciejewski, Frank Delaglio, Irina Bezsonova, and Jeffrey C Hoch. Nonuniform sampling in multidimensional NMR for improving spectral sensitivity. Methods, 138-9:62--68, 2018. [ DOI ]
The development of multidimensional NMR spectroscopy enabled an explosion of structural and dynamical investigations on proteins and other biomacromolecules. Practical limitations on data sampling, based on the Jeener paradigm of parametric sampling of indirect time domains, have long placed limits on resolution in the corresponding frequency dimensions. The emergence of nonuniform sampling (NUS) in indirect time dimensions circumvents those limitations, affording high resolution spectra from short data records collected in practically realized measurement times. In addition to substantially improved resolution, NUS can also be exploited to improve sensitivity, with gains comparable to those obtained using cryogenically cooled probes. We describe a general approach for acquiring and processing multidimensional NUS NMR data for improving sensitivity.
[52] Sergey Shnitkind, Maria A Martinez-Yamout, Jane Dyson, and Peter E Wright. Structural basis for graded inhibition of creb:dna interactions by multi-site phosphorylation. Biochemistry, 57(51):6964--6972, Dec 2018. [ DOI ]
Phosphorylation of the kinase-inducible domain (KID) of the cyclic AMP response element binding transcription factor (CREB) regulates its function through several mechanisms. Transcriptional activation occurs following phosphorylation at serine 133, but multisite phosphorylation in a neighboring region termed the CK cassette, residues 108-117, results in inhibition of CREB-mediated transcription. A molecular-level understanding of the mechanism of these opposing reactions has been lacking, in part because of the difficulty of preparing multiply-phosphorylated CREB in vitro. By substitution of a single residue, we have generated an engineered mammalian CREB in which the CK cassette can be phosphorylated in vitro by casein kinases and have characterized its interactions with cyclic AMP response element (CRE) DNA. Phosphorylation of the CK cassette promotes an intramolecular interaction between the KID domain and the site of DNA binding, the basic region of the C-terminal basic leucine zipper (bZip) domain. Competition between the phosphorylated KID domain and DNA for bZip binding results in lowered affinity of CREB for DNA. The binding free energy calculated from the dissociation constant is directly proportional to the number of phosphate groups in the CK cassette, indicating that the DNA binding is regulated by a rheostat-like mechanism. The rheostat is modulated by variation in the concentration of cations such as Mg2+ and by alternative isoforms such as the natural CREB isoform that lacks residues 162-272. Multisite phosphorylation of CREB represents a versatile mechanism by which transcription can be tuned to meet the variable needs of the cell.
[51] Scott A Robson, Koh Takeuchi, Andras Boeszoermenyi, Paul W Coote, Abhinav Dubey, Sven Hyberts, Gerhard Wagner, and Haribabu Arthanari. Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment. Nature Communications, 9(1):356, 01 2018. [ DOI ]
Backbone resonance assignment is a critical first step in the investigation of proteins by NMR. This is traditionally achieved with a standard set of experiments, most of which are not optimal for large proteins. Of these, HNCA is the most sensitive experiment that provides sequential correlations. However, this experiment suffers from chemical shift degeneracy problems during the assignment procedure. We present a strategy that increases the effective resolution of HNCA and enables near-complete resonance assignment using this single HNCA experiment. We utilize a combination of 2-13C and 3-13C pyruvate as the carbon source for isotope labeling, which suppresses the one bond (1Jx03B1x03B2) coupling providing enhanced resolution for the Cx03B1 resonance and amino acid-specific peak shapes that arise from the residual coupling. Using this approach, we can obtain near-complete (>85%) backbone resonance assignment of a 42x2009kDa protein using a single HNCA experiment.
[50] Joseph Masison, Paul J Michalski, Leslie M Loew, and Adam D Schuyler. mol2sphere: spherical decomposition of multi-domain molecules for visualization and coarse grained spatial modeling. Bioinformatics, 34(22):3948--3950, Nov 2018. [ DOI ]
Motivation: Proteins, especially those involved in signaling pathways are composed of functional modules connected by linker domains with varying degrees of flexibility. To understand the structure-function relationships in these macromolecules, it is helpful to visualize the geometric arrangement of domains. Furthermore, accurate spatial representation of domain structure is necessary for coarse-grain models of the multi-molecular interactions that comprise signaling pathways. Results: We introduce a new tool, mol2sphere, that transforms the atomistic structure of a macromolecule into a series of linked spheres corresponding to domains. It does this with a k-means clustering algorithm. It may be used for visualization or for coarse grain modeling and simulation. Availability and implementation: PyMOL plugin, source, and documentation. SpringSaLaD executables and documentation:, SpringSaLaD v.2 source:
[49] Mark W Maciejewski, Adam D Schuyler, and Jeffrey C Hoch. Practical nonuniform sampling and non-fourier spectral reconstruction for multidimensional NMR. In Protein NMR: Methods and Protocols, pages 341--352. Springer, 2018. [ DOI ]
A general approach to accelerating multidimensional NMR experiments via nonuniform sampling and maximum entropy spectral reconstruction was first demonstrated by Laue and colleagues in 1987. Following decades of continual improvements involving dozens of software packages for non-Fourier spectral analysis and many different schemes for nonuniform sampling, we still lack a clear consensus on best practices for sampling or spectral reconstruction, and programs for processing nonuniformly sampled data are not particularly user-friendly. Nevertheless, it is possible to discern conservative and general guidelines for nonuniform sampling and spectral reconstruction. Here, we describe a robust semi-automated workflow that employs these guidelines for simplifying the selection of a sampling schedule and the processing of the resulting nonuniformly sampled multidimensional NMR data. Our approach is based on NMRbox, a shared platform for NMR software that facilitates workflow development and execution, and enables rapid comparison of alternate approaches.
[48] Woonghee Lee and John L Markley. Pine-sparky.2 for automated nmr-based protein structure research. Bioinformatics, 34(9):1586--1588, 05 2018. [ DOI ]
Summary: Nuclear magnetic resonance (NMR) spectroscopy, along with X-ray crystallography and cryoelectron microscopy, is one of the three major tools that enable the determination of atomic-level structural models of biological macromolecules. Of these, NMR has the unique ability to follow important processes in solution, including conformational changes, internal dynamics and protein-ligand interactions. As a means for facilitating the handling and analysis of spectra involved in these types of NMR studies, we have developed PINE-SPARKY.2, a software package that integrates and automates discrete tasks that previously required interaction with separate software packages. The graphical user interface of PINE-SPARKY.2 simplifies chemical shift assignment and verification, automated detection of secondary structural elements, predictions of flexibility and hydrophobic cores, and calculation of three-dimensional structural models. Availability and implementation: PINE-SPARKY.2 is available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (, the NMRbox Project ( and to subscribers to the SBGrid ( For a detailed description of the program, see Contact: or Supplementary information: Supplementary data are available at Bioinformatics online.
[47] Priya Katyal, Yongkun Yang, You-Jun Fu, Jennifer Iandosca, Olga Vinogradova, and Yao Lin. Binding and backbone dynamics of protein under topological constraint: calmodulin as a model system. Chem Commun (Camb), 54(64):8917--8920, Aug 2018. [ DOI ]
Herein we present the effect of artificially imposed topological constraint on calmodulin (CaM) backbone dynamics and its molecular recognition behavior. While backbone dynamics of CaM remain largely unperturbed, the thermodynamic profile of CaM binding to the smooth-muscle myosin light-chain kinase (smMLCK) peptide is modulated significantly.
[46] Scott D Gorman, Debashish Sahu, Kathleen F O'Rourke, and David D Boehr. Assigning methyl resonances for protein solution-state nmr studies. Methods, 148:88--99, Sep 2018. [ DOI ]
Solution-state NMR is an important tool for studying protein structure and function. The ability to probe methyl groups has substantially expanded the scope of proteins accessible by NMR spectroscopy, including facilitating study of proteins and complexes greater than 100x202FkDa in size. While the toolset for studying protein structure and dynamics by NMR continues to grow, a major rate-limiting step in these studies is the initial resonance assignments, especially for larger (>50x202FkDa) proteins. In this practical review, we present strategies to efficiently isotopically label proteins, delineate NMR pulse sequences that can be used to determine methyl resonance assignments in the presence and absence of backbone assignments, and outline computational methods for NMR data analysis. We use our experiences from assigning methyl resonances for the aromatic biosynthetic enzymes tryptophan synthase and chorismate mutase to provide advice for all stages of experimental set-up and data analysis.
[45] Hesam Dashti, Jonathan R. Wedell, William M. Westler, Marco Tonelli, David Aceti, Gaya K. Amarasinghe, John L. Markley, and Hamid R. Eghbalnia. Applications of parametrized nmr spin systems of small molecules. Analytical Chemistry, 90(18):10646--10649, Aug 2018. [ DOI ]
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 1H 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
[44] Hesam Dashti, Jonathan Wedell, Gabriel Cornilescu, Charles Schwieters, William M Westler, John L Markley, and Hamid R Eghbalnia. Robust nomenclature and software for enhanced reproducibility in molecular modeling of small molecules. bioRxiv, 2018. [ DOI ]
Computational molecular dynamics, energy minimization, and modeling of molecular interactions are widely used in studies involving natural products, metabolites, and drugs. Manually directed computational steps commonly utilize an evolving collection of experimental and computational data, to which new data sources are added or modified as needed. Several software packages capable of incorporating sources of data are available, but the process remains error prone owing to the complexities of preparing and maintaining a consistent set of input files and the proper post-processing of derived data. We have devised a methodology and implemented it using an extensible software pipeline called RUNER (for Robust and Unique Nomenclature for Enhanced Reproducibility) that creates a robust and standardized computational process. The pipeline combines a web service and a graphical user interface (GUI) to enable seamless modifications and verified maintenance of atom force field parameters. The GUI provides an implementation for the widely used molecular modeling software package Xplor-NIH. We describe the RUNER software and demonstrate the rationale for the pipeline through examples of structural studies of small molecules and natural products. The software, pipeline, force field parameters, and file verification data for more than 4,100 compounds (including FDA-approved drugs and natural products) are freely accessible from [].
[43] Paul W Coote, Scott A Robson, Abhinav Dubey, Andras Boeszoermenyi, Mengxia Zhao, Gerhard Wagner, and Haribabu Arthanari. Optimal control theory enables homonuclear decoupling without Bloch-Siegert shifts in NMR spectroscopy. Nature Communications, 9(1):3014, 08 2018. [ DOI ]
The Bloch-Siegert shift is a phenomenon in NMR spectroscopy and atomic physics in which the observed resonance frequency is changed by the presence of an off-resonance applied field. In NMR, it occurs especially in the context of homonuclear decoupling. Here we develop a practical method for homonuclear decoupling that avoids inducing Bloch-Siegert shifts. This approach enables accurate observation of the resonance frequencies of decoupled nuclear spins. We apply this method to increase the resolution of the HNCA experiment. We also observe a doubling in sensitivity for a 30x2009kDa protein. We demonstrate the use of band-selective Cx03B2 decoupling to produce amino acid-specific line shapes, which are valuable for assigning resonances to the protein sequence. Finally, we assign the backbone of a 30 kDa protein, Human Carbonic Anhydrase II, using only HNCA experiments acquired with band-selective decoupling schemes, and instrument time of one week.
[42] Daiana A Capdevila, Fidel Huerta, Katherine A Edmonds, My Tra Le, Hongwei Wu, and David P Giedroc. Tuning site-specific dynamics to drive allosteric activation in a pneumococcal zinc uptake regulator. Elife, 7, 10 2018. [ DOI ]
MarR (multiple antibiotic resistance repressor) family proteins are bacterial repressors that regulate transcription in response to a wide range of chemical signals. Although specific features of MarR family function have been described, the role of atomic motions in MarRs remains unexplored thus limiting insights into the evolution of allostery in this ubiquitous family of repressors. Here, we provide the first experimental evidence that internal dynamics play a crucial functional role in MarR proteins. Streptococcus pneumoniae AdcR (adhesin-competence repressor) regulates ZnII homeostasis and ZnII functions as an allosteric activator of DNA binding. ZnII coordination triggers a transition from somewhat independent domains to a more compact structure. We identify residues that impact allosteric activation on the basis of ZnII-induced perturbations of atomic motions over a wide range of timescales. These findings appear to reconcile the distinct allosteric mechanisms proposed for other MarRs and highlight the importance of conformational dynamics in biological regulation.
[41] Kai Cai, Ronnie O. Frederick, Hesam Dashti, and John L. Markley. Architectural features of human mitochondrial cysteine desulfurase complexes from crosslinking mass spectrometry and small-angle x-ray scattering. Structure, 26(8):1127 -- 1136.e4, 2018. [ DOI ]
Cysteine desulfurase plays a central role in mitochondrial iron-sulfur cluster biogenesis by generating sulfur through the conversion of L-cysteine to L-alanine and by serving as the platform for assembling other components of the biosynthetic machinery, including ISCU, frataxin, and ferredoxin. The human mitochondrial cysteine desulfurase complex consists of two copies each of NFS1, ISD11, and acyl carrier protein. We describe results from chemical crosslinking coupled with tandem mass spectrometry and small-angle X-ray scattering studies that are consistent with a closed NFS1 dimer rather than an open one for both the cysteine desulfurase-ISCU and cysteine desulfurase-ISCU-frataxin complexes. We present a structural model for the cysteine desulfurase-ISCU-frataxin complex derived from chemical crosslinking restraints in conjunction with the recent crystal structure of the cysteine desulfurase-ISCU-zinc complex and distance constraints from nuclear magnetic resonance.
[40] Michael R Gryk and Bertram Ludäscher. Semantic mediation to improve reproducibility for biomolecular NMR analysis. Transform Digit Worlds (2018), 10766:620--625, Mar 2018. [ DOI ]
Two barriers to computational reproducibility are the ability to record the critical metadata required for rerunning a computation, as well as translating the semantics of the metadata so that alternate approaches can easily be configured for verifying computational reproducibility. We are addressing this problem in the context of biomolecular NMR computational analysis by developing a series of linked ontologies which define the semantics of the various software tools used by researchers for data transformation and analysis. Building from a core ontology representing the primary observational data of NMR, the linked data approach allows for the translation of metadata in order to configure alternate software approaches for given computational tasks. In this paper we illustrate the utility of this with a small sample of the core ontology as well as tool-specific semantics for two third-party software tools. This approach to semantic mediation will help support an automated approach to validating the reliability of computation in which the same processing workflow is implemented with different software tools. In addition, the detailed semantics of both the data and the processing functionalities will provide a method for software tool classification.
[39] Khiem Q Nguyen, Elizabeth Zecca, Olga Vinogradova, and Devendra S Kalonia. NMR as a semi-quantitative tool for evaluating protein surface hydrophobicity. SciFed Pharmaceutics Journal, 2018. [ http ]
Nuclear Magnetic Resonance spectroscopy was applied for evaluation of the surface hydrophobicity of three proteins, Bovine Serum Albumin, α-chymotrypsinogen A and β-lactoglobulin A. The protein surface hydrophobicity was correlated with the binding of small molecular probes selected based upon their aliphatic or aromatic moieties. The interactions were quantified through the transverse relaxation time, T2, where a significant decrease in the transverse relaxation time of the small probe indicated a more pronounced hydrophobic binding to the protein. For all proteins, phenol, an aromatic alcohol, acted as the most informative probe and showed that BSA is the most hydrophobic of proteins studied. The comparison between A-ChytA and B-LgA came inconclusive due the severe instability of A-ChtA in solution. In addition, more common classical approaches for surface hydrophobicity evaluation, HIC and fluorescence spectroscopy, were in agreement with the NMR results. For HIC experiments in particular, two columns were used to further assess the difference in aromatic and aliphatic interactions, confirming the notion of prevalent aromatic binding for all three proteins.
[38] Britta E. Rued, Martín Alcorlo, Katherine A. Edmonds, Siseth Martínez-Caballero, Daniel Straume, Yue Fu, Kevin E. Bruce, Hongwei Wu, Leiv Sigve Håvarstein, Juan A. Hermoso, Malcolm E. Winkler, and David P. Giedroc. Structure of the large extracellular loop of ftsx and its interaction with the essential peptidoglycan hydrolase pcsb in streptococcus pneumoniae. bioRxiv, 2018. [ DOI ]
Streptococcus pneumoniae is a leading killer of infants and immunocompromised adults and has become increasingly resistant to major antibiotics. Therefore, the development of new antibiotic strategies is desperately needed. Targeting bacterial cell division is one such strategy, specifically targeting essential proteins for the synthesis and breakdown of peptidoglycan. One complex important to this process is FtsEX. FtsEX comprises an integral membrane protein (FtsX) and cytoplasmic ATPase (FtsE) that resembles an ATP-binding cassette (ABC) transporter. Here, we present NMR solution structural and crystallographic models of the large extracellular domain of FtsX, denoted ECL1. The structure of ECL1 reveals an upper extended β-hairpin and a lower α-helical lobe, each extending from a mixed α-β core. The helical lobe mediates a physical interaction with the peptidoglycan hydrolase PcsB, via the coiled-coil domain of PcsB (PcsB-CC). Characterization of S. pneumoniae D39 derived strains harboring mutations in the α-helical lobe shows that this subdomain is essential for cell viability and required for proper cell division of S. pneumoniae.
[37] John L. Markley. Current solution NMR techniques for structure-function studies of proteins and RNA molecules. Integrative Structural Biology with Hybrid Methods, pages 43--58, 2018. [ DOI ]
We briefly review current technology for structure-function investigations of biological macromolecules in solution by nuclear magnetic resonance spectroscopy, which enable hybrid methods. An advantage of NMR is that biomolecules can be studied at atomic resolution under near physiological conditions where they are dynamically active. We outline stable isotope labeling strategies, NMR data collection methodology, and procedures for data analysis leading to structure-function information. We discuss issues related to NMR software and data deposition.
[36] Rebecca L. Newcomer, Helen B. Belato, Carolyn M. Teschke, and Andrei T. Alexandrescu. NMR assignments for monomeric phage L decoration protein. Biomolecular NMR Assignments, 12(2):339--343, Aug 2018. [ DOI ]
Phage L encodes a trimeric 43 kDa decoration protein (Dec) that noncovalently binds and stabilizes the capsids of the homologous phages L and P22 in vitro. At physiological pH Dec was unsuitable for NMR. We were able to obtain samples amenable for NMR spectroscopy by unfolding Dec to pH 2 and refolding it to pH 4. Our unfolding/refolding protocol converted trimeric Dec to a folded 14.4 kDa monomer. We verified that the acid-unfolding protocol did not perturb the secondary structure, or the capsid-binding function of refolded Dec. We were able to obtain complete 1H, 15N, and 13C assignments for the Dec monomer, as well as information on its secondary structure and dynamics based on chemical shift assignments. The assigned NMR spectrum is being used to determine the three-dimensional structure of Dec, which is important for understanding how the trimer binds phage capsids and for the use of the protein as a platform for phage-display nanotechnology.
[35] Eldon L. Ulrich, Kumaran Baskaran, Hesam Dashti, Yannis E. Ioannidis, Miron Livny, Pedro R. Romero, Dimitri Maziuk, Jonathan R. Wedell, Hongyang Yao, Hamid R. Eghbalnia, and et al. NMR-STAR: comprehensive ontology for representing, archiving and exchanging data from nuclear magnetic resonance spectroscopic experiments. Journal of Biomolecular NMR, Dec 2018. [ DOI ]
The growth of the biological nuclear magnetic resonance (NMR) field and the development of new experimental technology have mandated the revision and enlargement of the NMR-STAR ontology used to represent experiments, spectral and derived data, and supporting metadata. We present here a brief description of the NMR-STAR ontology and software tools for manipulating NMR-STAR data files, editing the files, extracting selected data, and creating data visualizations. Detailed information on these is accessible from the links provided.
[34] Chris Morris. The life cycle of structural biology data. Data Science Journal, 17, 2018. [ DOI ]
Research data is acquired, interpreted, published, reused, and sometimes eventually discarded. Understanding this life cycle better will help the development of appropriate infrastructural services, ones which make it easier for researchers to preserve, share, and find data. Structural biology is a discipline within the life sciences, one that investigates the molecular basis of life by discovering and interpreting the shapes and motions of macromolecules. Structural biology has a strong tradition of data sharing, expressed by the founding of the Protein Data Bank (PDB) in 1971. The culture of structural biology is therefore already in line with the perspective that data from publicly funded research projects are public data. This review is based on the data life cycle as defined by the UK Data Archive. It identifies six stages: creating data, processing data, analysing data, preserving data, giving access to data, and re-using data. For clarity, ʻpreserving dataʼ and ʻgiving access to dataʼ are discussed together. A final stage to the life cycle, ʻdiscarding dataʼ, is also discussed. The review concludes with recommendations for future improvements to the IT infrastructure for structural biology.
[33] Dillon B. Nye and Juliette T. J. Lecomte. Replacement of the distal histidine reveals a noncanonical heme binding site in a 2-on-2 hemoglobin. Biochemistry, 57(40):5785--5796, Sep 2018. [ DOI | http ]
Heme ligation in hemoglobin is typically assumed by the “proximal” histidine. Hydrophobic contacts, ionic interactions, and the ligation bond secure the heme between two α-helices denoted E and F. Across the hemoglobin superfamily, several proteins also use a “distal” histidine, making the native state a bis-histidine complex. The group 1 truncated hemoglobin from Synechocystis sp. PCC 6803, GlbN, is one such bis-histidine protein. Ferric GlbN, in which the distal histidine (His46 or E10) has been replaced with a leucine, though expected to bind a water molecule and yield a high-spin iron complex at neutral pH, has low-spin spectral properties. Here, we applied nuclear magnetic resonance and electronic absorption spectroscopic methods to GlbN modified with heme and amino acid replacements to identify the distal ligand in H46L GlbN. We found that His117, a residue located in the C-terminal portion of the protein and on the proximal side of the heme, is responsible for the formation of an alternative bis-histidine complex. Simultaneous coordination by His70 and His117 situates the heme in a binding site different from the canonical site. This new holoprotein form is achieved with only local conformational changes. Heme affinity in the alternative site is weaker than in the normal site, likely because of strained coordination and a reduced number of specific heme--protein interactions. The observation of an unconventional heme binding site has important implications for the interpretation of mutagenesis results and globin homology modeling.
[32] Biswapriya B. Misra and Subhashree Mohapatra. Tools and resources for metabolomics research community: A 2017-2018 update. ELECTROPHORESIS, Nov 2018. [ DOI ]
The scale at which MS‐ and NMR‐based platforms generate metabolomics datasets for both research, core, and clinical facilities to address challenges in the various sciences---ranging from biomedical to agricultural---is underappreciated. Thus, metabolomics efforts spanning microbe, environment, plant, animal, and human systems have led to continual and concomitant growth of in silico resources for analysis and interpretation of these datasets. These software tools, resources, and databases drive the field forward to help keep pace with the amount of data being generated and the sophisticated and diverse analytical platforms that are being used to generate these metabolomics datasets. To address challenges in data preprocessing, metabolite annotation, statistical interrogation, visualization, interpretation, and integration, the metabolomics and informatics research community comes up with hundreds of tools every year. The purpose of the present review is to provide a brief and useful summary of more than 95 metabolomics tools, software, and databases that were either developed or significantly improved during 2017--2018. We hope to see this review help readers, developers, and researchers to obtain informed access to these thorough lists of resources for further improvisation, implementation, and application in due course of time.
[31] Hesam Dashti, William M. Westler, John L. Markley, and Hamid R. Eghbalnia. Unique identifiers for small molecules enable rigorous labeling of their atoms. Scientific Data, 4:170073, May 2017. [ DOI ]
Rigorous characterization of small organic molecules in terms of their structural and biological properties is vital to biomedical research. The three-dimensional structure of a molecule, its `photo ID', is inefficient for searching and matching tasks. Instead, identifiers play a key role in accessing compound data. Unique and reproducible molecule and atom identifiers are required to ensure the correct cross-referencing of properties associated with compounds archived in databases. The best approach to this requirement is the International Chemical Identifier (InChI). However, the current implementation of InChI fails to provide a complete standard for atom nomenclature, and incorrect use of the InChI standard has resulted in the proliferation of non-unique identifiers. We propose a methodology and associated software tools, named ALATIS, that overcomes these shortcomings. ALATIS is an adaptation of InChI, which operates fully within the InChI convention to provide unique and reproducible molecule and all atom identifiers. ALATIS includes an InChI extension for unique atom labeling of symmetric molecules. ALATIS forms the basis for improving reproducibility and unifying cross-referencing across databases.
[30] Rebekah R Shippy, Xiaochen Lin, Sherry S Agabiti, Jin Li, Brendan M Zangari, Benjamin J Foust, Michael M Poe, Chia-Hung Christine Hsiao, Olga Vinogradova, David F Wiemer, and Andrew J Wiemer. Phosphinophosphonates and their tris-pivaloyloxymethyl prodrugs reveal a negatively cooperative butyrophilin activation mechanism. J Med Chem, 60(6):2373--2382, 03 2017. [ DOI ]
Butyrophilin 3A1 (BTN3A1) binds small phosphorus-containing molecules, which initiates transmembrane signaling and activates butyrophilin-responsive cells. We synthesized several phosphinophosphonates and their corresponding tris-pivaloyloxymethyl (tris-POM) prodrugs and examined their effects on BTN3A1. An analog of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) bound to BTN3A1 with intermediate affinity, which was enthalpy-driven. Docking studies revealed binding to the basic surface pocket and interactions between the allylic hydroxyl group and the BTN3A1 backbone. The phosphinophosphonate stimulated proliferation of Vx03B39Vx03B42 T cells with moderate activity (EC50 = 26 x03BCM). Cellular potency was enhanced >600-fold in the tris-POM prodrug (EC50 = 0.041 x03BCM). The novel prodrug also induced T cell mediated leukemia cell lysis. Analysis of dose-response data reveals HMBPP-induced Hill coefficients of 0.69 for target cell lysis and 0.68 in interferon secretion. Together, tris-POM prodrugs enhance the cellular activity of phosphinophosphonates, reveal structure-activity relationships of butyrophilin ligands, and support a negatively cooperative model of cellular butyrophilin activation.
[29] Robbins Puthenveetil, Khiem Nguyen, and Olga Vinogradova. Nanodiscs and solution nmr: preparation, application and challenges. Nanotechnol Rev, 6(1):111--126, Feb 2017. [ DOI ]
Nanodiscs provide an excellent system for the structure-function investigation of membrane proteins. Its direct advantage lies in presenting a water soluble form of an otherwise hydrophobic molecule, making it amenable to a plethora of solution techniques. Nuclear Magnetic Resonance is one such high resolution approach that looks at the structure and dynamics of a protein with atomic level precision. Recently, there has been a breakthrough in making nanodiscs more susceptible for structure determination by solution NMR, yet it still remains to become the preferred choice for a membrane mimetic. In this practical review, we provide a general discourse on nanodisc and its application to solution NMR. We also offer potential solutions to remediate the technical challenges associated with nanodisc preparation and the choice of proper experimental set-ups. Along with discussing several structural applications, we demonstrate an alternative use of nanodiscs for functional studies, where we investigated the phosphorylation of a cell surface receptor, Integrin. This is the first successful manifestation of observing activated receptor phosphorylation in nanodiscs through NMR. We additionally present an on-column method for nanodisc preparation with multiple strategies and discuss the potential use of alternative nanoscale phospholipid bilayer systems like SMA lipid discs and Saposin-A lipoprotein discs.
[28] Robbins Puthenveetil, Sanjiv Kumar, Melissa J Caimano, Abhishek Dey, Arvind Anand, Olga Vinogradova, and Justin D Radolf. The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of treponema denticola. Sci Rep, 7(1):13260, Oct 2017. [ DOI ]
The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSPN and MOSPC. Biophysical analysis of constructs refolded in vitro demonstrated that MOSPC, previously shown to possess porin activity, forms amphiphilic trimers, while MOSPN forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSPC is OM-embedded and surface-exposed, while MOSPN resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the x03B2-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.
[27] Khiem Nguyen, Robbins Puthenveetil, and Olga Vinogradova. Investigation of the adaptor protein PLIC-2 in multiple pathways. Biochem Biophys Rep, 9:341--348, Mar 2017. [ DOI ]
PLIC, Protein Linking IAP (CD47) to Cytoskeleton, have long since been implicated in connecting the extracellular membrane to the intracellular cell cytoskeleton. This phenomenon is supposedly achieved by bridging a receptor protein CD47 to vimentin, an intermediate filament, which in turn regulates integrin dependent cell spreading. Since the discovery of these proteins, the molecular details of the above-mentioned interactions and the underlying complexes are yet to be characterized. Several independent studies have together emphasized PLIC/Ubiquilin's role in the proteasomal degradation pathway. This seems to be in contrast to the purported initial discovery of PLIC as a cytoskeletal adaptor protein. In an effort to reconcile the different roles associated with the ubiquitous PLIC proteins, we tested the involvement of PLIC-2 both in the proteasomal degradation pathway and as a protein linking the cell cytoskeleton to the cytoplasmic tail of CD47. This was achieved thorough an in vitro investigation of their binding interface using a combination of biophysical techniques. Our results show that the two terminal domains of PLIC-2 interact weakly with each other, while the C-terminal UBA domain interacts strongly with ubiquitin. Interestingly, no perceptible interaction was observed for PLIC-2 with the cytoplasmic tail of CD47 questioning its role as a "PLIC" protein linking the cell membrane to the cytoskeleton.
[26] Hatef Monajemi, David L. Donoho, Jeffrey C. Hoch, and Adam D. Schuyler. Incoherence of partial-component sampling in multidimensional nmr. arXiv, 02 2017. [ arXiv ]
In NMR spectroscopy, undersampling in the indirect dimensions causes reconstruction artifacts whose size can be bounded using the so-called coherence. In experiments with multiple indirect dimensions, new undersampling approaches were recently proposed: random phase detection (RPD) [?] and its generalization, partial component sampling (PCS) [?]. The new approaches are fully aware of the fact that high-dimensional experiments generate hypercomplex-valued free induction decays; they randomly acquire only certain low-dimensional components of each high-dimensional hypercomplex entry. We provide a classification of various hypercomplex-aware undersampling schemes, and define a hypercomplex-aware coherence appropriate for such undersampling schemes; we then use it to quantify undersampling artifacts of RPD and various PCS schemes.
[25] Mark W Maciejewski, Adam D Schuyler, Michael R Gryk, Ion I Moraru, Pedro R Romero, Eldon L Ulrich, Hamid R Eghbalnia, Miron Livny, Frank Delaglio, and Jeffrey C Hoch. NMRbox: A resource for biomolecular NMR computation. Biophysical Journal, 112(8):1529--1534, 2017. [ DOI ]
Advances in computation have been enabling many recent advances in biomolecular applications of NMR. Due to the wide diversity of applications of NMR, the number and variety of software packages for processing and analyzing NMR data is quite large, with labs relying on dozens, if not hundreds of software packages. Discovery, acquisition, installation, and maintenance of all these packages is a burdensome task. Because the majority of software packages originate in academic labs, persistence of the software is compromised when developers graduate, funding ceases, or investigators turn to other projects. To simplify access to and use of biomolecular NMR software, foster persistence, and enhance reproducibility of computational workflows, we have developed NMRbox, a shared resource for NMR software and computation. NMRbox employs virtualization to provide a comprehensive software environment preconfigured with hundreds of software packages, available as a downloadable virtual machine or as a Platform-as-a-Service supported by a dedicated compute cloud. Ongoing development includes a metadata harvester to regularize, annotate, and preserve workflows and facilitate and enhance data depositions to BioMagResBank, and tools for Bayesian inference to enhance the robustness and extensibility of computational analyses. In addition to facilitating use and preservation of the rich and dynamic software environment for biomolecular NMR, NMRbox fosters the development and deployment of a new class of metasoftware packages. NMRbox is freely available to not-for-profit users.
[24] Priya Katyal, Yongkun Yang, Olga Vinogradova, and Yao Lin. Expression of cellulolytic enzyme as a fusion protein that reacts specifically with a polymeric scaffold. Methods Enzymol, 590:259--276, 2017. [ DOI ]
The formation of higher-order assemblies of multiple proteins or enzymes is a general mechanism to achieve more sophisticated biological function in biological systems. For example, cellulosomes are large complexes consisting of multiple cellulolytic enzymes that rely on the concerted actions of different enzymes built onto a common protein scaffold to facilitate the breakdown of the polymeric substrate, cellulose. One strategy for mimicking these highly effective nanomachines may involve the use of synthetic scaffolds that can react to and organize multiple engineered enzymes to promote synergistic action between the enzymes on the scaffold. As an example of the earlier strategy, we describe here an approach for the expression of cellulolytic enzymes with a serine esterase tag, and the rapid reaction between the tag and the end-functionalized polymers to form enzyme-polymer-enzyme multienzyme conjugates. In principle, this general and versatile supramolecular approach may be used to organize specific cellulolytic enzymes onto synthetic scaffolds to form multienzyme complexes to potentially work in synergy for enhanced biological activities. Best reaction conditions, good activities of the armored cellulolytic enzymes and the design of optimal protein linker in the fusion protein are discussed in detail. If other reactive tags are included on the enzyme in future, multiple types of synergistic enzymes may be positioned at specific sites on a designed polymer scaffold that mimics the complex structure and enhanced function of natural cellulosomes. This type of nanoarmoring of multiple enzymes on a nanoscale might also enhance enzyme stability, when compared to the unprotected enzymes.
[23] Michael R. Gryk and Bertram Ludäscher. Workflows and provenance: Toward information science solutions for the natural sciences. Library Trends, 65(4):555--562, 2017. [ DOI ]
The era of big data and ubiquitous computation has brought with it concerns about ensuring reproducibility in this new research environment. It is easy to assume that computational methods self-document by their very nature of being exact, deterministic processes. However, similar to laboratory experiments, ensuring reproducibility in the computational realm requires the documentation of both the protocols used (workflows), as well as a detailed description of the computational environment: algorithms, implementations, software environments, and the data ingested and execution logs of the computation. These two aspects of computational reproducibility (workflows and execution details) are discussed within the context of biomolecular Nuclear Magnetic Resonance spectroscopy (bioNMR), as well as the PRIMAD model for computational reproducibility.
[22] Hesam Dashti, William M Westler, Marco Tonelli, Jonathan R Wedell, John L Markley, and Hamid R Eghbalnia. Spin system modeling of nuclear magnetic resonance spectra for applications in metabolomics and small molecule screening. Anal Chem, 89(22):12201--12208, Nov 2017. [ DOI ]
The exceptionally rich information content of nuclear magnetic resonance (NMR) spectra is routinely used to identify and characterize molecules and molecular interactions in a wide range of applications, including clinical biomarker discovery, drug discovery, environmental chemistry, and metabolomics. The set of peak positions and intensities from a reference NMR spectrum generally serves as the identifying signature for a compound. Reference spectra normally are collected under specific conditions of pH, temperature, and magnetic field strength, because changes in conditions can distort the identifying signatures of compounds. A spin system matrix that parametrizes chemical shifts and coupling constants among spins provides a much richer feature set for a compound than a spectral signature based on peak positions and intensities. Spin system matrices expand the applicability of NMR spectral libraries beyond the specific conditions under which data were collected. In addition to being able to simulate spectra at any field strength, spin parameters can be adjusted to systematically explore alterations in chemical shift patterns due to variations in other experimental conditions, such as compound concentration, pH, or temperature. We present methodology and software for efficient interactive optimization of spin parameters against experimental 1D-1H NMR spectra of small molecules. We have used the software to generate spin system matrices for a set of key mammalian metabolites and are also using the software to parametrize spectra of small molecules used in NMR-based ligand screening. The software, along with optimized spin system matrix data for a growing number of compounds, is available from .
[21] Frank Delaglio, Gregory S Walker, Kathleen A Farley, Raman Sharma, Jeffrey C Hoch, Luke W Arbogast, Robert G Brinson, and John P Marino. Non-uniform sampling for all: More NMR spectral quality, less measurement time. American pharmaceutical review, 20(4), 2017.
No abstract.
[20] Hamid R Eghbalnia, Pedro R Romero, William M Westler, Kumaran Baskaran, Eldon L Ulrich, and John L Markley. Increasing rigor in nmr-based metabolomics through validated and open source tools. Curr Opin Biotechnol, 43:56--61, 02 2017. [ DOI ]
The metabolome, the collection of small molecules associated with an organism, is a growing subject of inquiry, with the data utilized for data-intensive systems biology, disease diagnostics, biomarker discovery, and the broader characterization of small molecules in mixtures. Owing to their close proximity to the functional endpoints that govern an organism's phenotype, metabolites are highly informative about functional states. The field of metabolomics identifies and quantifies endogenous and exogenous metabolites in biological samples. Information acquired from nuclear magnetic spectroscopy (NMR), mass spectrometry (MS), and the published literature, as processed by statistical approaches, are driving increasingly wider applications of metabolomics. This review focuses on the role of databases and software tools in advancing the rigor, robustness, reproducibility, and validation of metabolomics studies.
[19] John L Markley, Rafael Brüschweiler, Arthur S Edison, Hamid R Eghbalnia, Robert Powers, Daniel Raftery, and David S Wishart. The future of NMR-based metabolomics. Current opinion in biotechnology, 43:34--40, 02 2017. [ DOI ]
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.
[18] Jeffrey C Hoch. Beyond Fourier. J Magn Reson, 283:117--123, Oct 2017. [ DOI ]
Non-Fourier methods of spectrum analysis are gaining traction in NMR spectroscopy, driven by their utility for processing nonuniformly sampled data. These methods afford new opportunities for optimizing experiment time, resolution, and sensitivity of multidimensional NMR experiments, but they also pose significant challenges not encountered with the discrete Fourier transform. A brief history of non-Fourier methods in NMR serves to place different approaches in context. Non-Fourier methods reflect broader trends in the growing importance of computation in NMR, and offer insights for future software development.
[17] Matthew A Zambrello, Mark W Maciejewski, Adam D Schuyler, Gerard Weatherby, and Jeffrey C Hoch. Robust and transferable quantification of NMR spectral quality using IROC analysis. J Magn Reson, 285:37--46, Dec 2017. [ DOI ]
Non-Fourier methods are increasingly utilized in NMR spectroscopy because of their ability to handle nonuniformly-sampled data. However, non-Fourier methods present unique challenges due to their nonlinearity, which can produce nonrandom noise and render conventional metrics for spectral quality such as signal-to-noise ratio unreliable. The lack of robust and transferable metrics (i.e. applicable to methods exhibiting different nonlinearities) has hampered comparison of non-Fourier methods and nonuniform sampling schemes, preventing the identification of best practices. We describe a novel method, in situ receiver operating characteristic analysis (IROC), for characterizing spectral quality based on the Receiver Operating Characteristic curve. IROC utilizes synthetic signals added to empirical data as "ground truth", and provides several robust scalar-valued metrics for spectral quality. This approach avoids problems posed by nonlinear spectral estimates, and provides a versatile quantitative means of characterizing many aspects of spectral quality. We demonstrate applications to parameter optimization in Fourier and non-Fourier spectral estimation, critical comparison of different methods for spectrum analysis, and optimization of nonuniform sampling schemes. The approach will accelerate the discovery of optimal approaches to nonuniform sampling experiment design and non-Fourier spectrum analysis for multidimensional NMR.
[16] Khiem Nguyen, Jin Li, Robbins Puthenveetil, Xiaochen Lin, Michael M. Poe, Chia-Hung Christine Hsiao, Olga Vinogradova, and Andrew J. Wiemer. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region. The FASEB Journal, 31(11):4697--4706, Nov 2017. [ DOI ]
[15] Anne R. Kaplan, Katherine Kaus, Swastik De, Rich Olson, and Andrei T. Alexandrescu. NMR structure of the Bacillus cereus hemolysin ii C-terminal domain reveals a novel fold. Scientific Reports, 7(1), Jun 2017. [ DOI ]
In addition to multiple virulence factors, Bacillus cereus a pathogen that causes food poisoning and life-threatening wound infections, secretes the pore-forming toxin hemolysin II (HlyII). The HlyII toxin has a unique 94 amino acid C-terminal domain (HlyIIC). HlyIIC exhibits splitting of NMR resonances due to cis/trans isomerization of a single proline near the C-terminus. To overcome heterogeneity, we solved the structure of P405M-HlyIIC, a mutant that exclusively stabilizes the trans state. The NMR structure of HlyIIC reveals a novel fold, consisting of two subdomains αA-β1-β2 and β3-β4-αB-β5, that come together in a barrel-like structure. The barrel core is fastened by three layers of hydrophobic residues. The barrel end opposite the HlyIIC-core has a positively charged surface, that by binding negatively charged moieties on cellular membranes, may play a role in target-cell surface recognition or stabilization of the heptameric pore complex. In the WT domain, dynamic flexibility occurs at the N-terminus and the first α-helix that connects the HlyIIC domain to the HlyII-core structure. In the destabilizing P405M mutant, increased flexibility is evident throughout the first subdomain, suggesting that the HlyIIC structure may have arisen through gene fusion.
[14] Anne R. Kaplan, Megan R. Brady, Mark W. Maciejewski, Richard A. Kammerer, and Andrei T. Alexandrescu. Nuclear magnetic resonance structures of GCN4p are largely conserved when ion pairs are disrupted at acidic pH but show a relaxation of the coiled coil superhelix. Biochemistry, 56(11):1604--1619, Mar 2017. [ DOI ]
To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C′, Cα, and Cβ) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of 1H--15N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, 13C rotating frame T1 relaxation (T1ρ) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.
[13] Bruce A. Johnson. From raw data to protein backbone chemical shifts using NMRFx processing and NMRViewJ analysis. Protein NMR, pages 257--310, Nov 2017. [ DOI ]
Assignment of the chemical shifts of the backbone atoms (HN, N, CA, CB, and C) of proteins is often a prerequisite to using NMR information in the study of proteins. These chemical shifts and their perturbations are the basis for the analysis of protein dynamics, ligand binding, and backbone conformation. They are generally assigned prior to full side-chain assignments and the determination of the complete three-dimensional molecular structure. This chapter describes the use of two software packages, NMRFx Processor and NMRViewJ, in going from raw NMR data to backbone assignments. The step-by-step procedure describes processing of the data and the use of manual and automated features of the RunAbout tool in NMRViewJ to perform the assignments.
[12] Mehdi Mobli and Jeffrey C Hoch. Fast NMR Data Acquisition: Beyond the Fourier Transform. Royal Society of Chemistry, 2017.
No abstract.
[11] Mehdi Mobli, Eivind A.B. Undheim, and Lachlan D. Rash. Chapter seven - modulation of ion channels by cysteine-rich peptides: From sequence to structure. In Dominic P. Geraghty and Lachlan D. Rash, editors, Ion Channels DownUnder, volume 79 of Advances in Pharmacology, pages 199 -- 223. Academic Press, 2017. [ DOI ]
Venom peptides are natural ligands of ion channels and have been used extensively in pharmacological characterization of various ion channels and receptors. In this chapter, we survey all known venom peptide ion-channel modulators. Our survey reveals that the majority of venom peptides characterized to date target voltage-gated sodium or potassium channels. We further find that the majority of these peptides are found in scorpion and spider venoms. We discuss the influence of the pharmacological tools available in biasing discovery and the classical “toxin-to-sequence” approach to venom peptide biodiscovery. The impact of high-throughput sequencing on the existing discovery framework is likely to be significant and we propose here an alternative “sequence-to-toxin” approach to peptide screening, relying more on recently developed high-throughput methods. Methods for production and characterization of disulfide rich toxins in a high-throughput setting are then described, focusing on bacterial protein expression and solution state structural characterization by NMR spectroscopy. Finally, the role of X-ray crystallography and cryo-EM are highlighted by discussing the currently known channel-peptide complexes.
[10] John L. Markley and William Milo Westler. Biomolecular NMR: Past and future. Archives of Biochemistry and Biophysics, 628:3 -- 16, 2017. Nuclear Magnetic Resonance. [ DOI ]
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 NMR, and our brief personal account certainly omits many important milestones.
[9] Jia Xu and Steven R. Van Doren. Tracking equilibrium and nonequilibrium shifts in data with TREND. Biophysical Journal, 112(2):224 -- 233, 2017. [ DOI ]
Principal component analysis (PCA) discovers patterns in multivariate data that include spectra, microscopy, and other biophysical measurements. Direct application of PCA to crowded spectra, images, and movies (without selecting peaks or features) was shown recently to identify their equilibrium or temporal changes. To enable the community to utilize these capabilities with a wide range of measurements, we have developed multiplatform software named TREND to Track Equilibrium and Nonequilibrium population shifts among two-dimensional Data frames. TREND can also carry this out by independent component analysis. We highlight a few examples of finding concurrent processes. TREND extracts dual phases of binding to two sites directly from the NMR spectra of the titrations. In a cardiac movie from magnetic resonance imaging, TREND resolves principal components (PCs) representing breathing and the cardiac cycle. TREND can also reconstruct the series of measurements from selected PCs, as illustrated for a biphasic, NMR-detected titration and the cardiac MRI movie. Fidelity of reconstruction of series of NMR spectra or images requires more PCs than needed to plot the largest population shifts. TREND reads spectra from many spectroscopies in the most common formats (JCAMP-DX and NMR) and multiple movie formats. The TREND package thus provides convenient tools to resolve the processes recorded by diverse biophysical methods.
[8] Mark Bostock and Daniel Nietlispach. Compressed sensing: Reconstruction of non-uniformly sampled multidimensional NMR data. Concepts in Magnetic Resonance Part A, 46A(2):e21438, Mar 2017. [ DOI ]
Nuclear magnetic resonance (NMR) spectroscopy is widely used across the physical, chemical, and biological sciences. A core component of NMR studies is multidimensional experiments, which enable correlation of properties from one or more NMR‐active nuclei. In high‐resolution biomolecular NMR, common nuclei are 1H, 15N, and 13C, and triple resonance experiments using these three nuclei form the backbone of NMR structural studies. In other fields, a range of other nuclei may be used. Multidimensional NMR experiments provide unparalleled information content, but this comes at the price of long experiment times required to achieve the necessary resolution and sensitivity. Non‐uniform sampling (NUS) techniques to reduce the required data sampling have existed for many decades. Recently, such techniques have received heightened interest due to the development of compressed sensing (CS) methods for reconstructing spectra from such NUS datasets. When applied jointly, these methods provide a powerful approach to dramatically improve the resolution of spectra per time unit and under suitable conditions can also lead to signal‐to‐noise ratio improvements. In this review, we explore the basis of NUS approaches, the fundamental features of NUS reconstruction using CS and applications based on CS approaches including the benefits of expanding the repertoire of biomolecular NMR experiments into higher dimensions. We discuss some of the recent algorithms and software packages and provide practical tips for recording and processing NUS data by CS.
[7] Fariba Fathi, Antonio Brun, Katherine Rott, Paulo Falco Cobra, Marco Tonelli, Hamid Eghbalnia, Enrique Caviedes-Vidal, William Karasov, and John Markley. NMR-based identification of metabolites in polar and non-polar extracts of avian liver. Metabolites, 7(4):61, Nov 2017. [ DOI ]
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 the detection of 52 polar and nine non-polar metabolites in 1H NMR spectra of the extracts. We analyzed the lipophilic metabolites by one-way ANOVA to assess statistically significant concentration differences between the two groups. The results of our studies demonstrate that the protocol described here can be exploited for high-throughput screening of small quantities of liver tissue (approx. 100 mg wet mass) obtainable from small animals.
[6] Mark W Maciejewski, Adam D Schuyler, Michael R Gryk, Ion I Moraru, Pedro R Romero, Hamid R Eghbalnia, Miron Livny, Frank Delaglio, and Jeffrey C Hoch. RE: another barrier to reproducibility, an eLetter response to "Enhancing reproducibility for computational methods". Science, 354(6317):1240--1241, 12 2016. [ DOI ]
No abstract.
[5] Therese N. Tripler, Carolyn M. Teschke, and Andrei T. Alexandrescu. NMR assignments for the insertion domain of bacteriophage Sf6 coat protein. Biomolecular NMR Assignments, 11(1):35--38, Oct 2016. [ DOI ]
The P22 bacteriophage group is a subgroup of the λ phage supercluster, comprised of the three major sequence types Sf6, P22, and CUS-3, based on their capsid proteins. Our goal is to investigate the extent to which structure--function relationships are conserved for the viral coat proteins and I-domains in this subgroup. Sf6 is a phage that infects the human pathogen Shigella flexneri. The coat protein of Sf6 assembles into a procapsid, which further undergoes maturation during DNA packaging into an infectious virion. The Sf6 coat protein contains a genetically inserted domain, termed the I-domain, similar to the ones present in the P22 and CUS-3 coat proteins. Based on the P22 example, I-domains play important functional roles in capsid assembly, stability, viability, and size-determination. Here we report the 1H, 15N, and 13C chemical shift assignments for the I-domain of the Sf6 phage coat protein. Chemical shift-based secondary structure prediction and hydrogen-bond patterns from a long-range HNCO experiment indicate that the Sf6 I-domain adopts a 6-stranded β-barrel fold like those of P22 and CUS-3 but with important differences, including the absence of the D-loop that is critical for capsid assembly and the addition of a novel disordered loop region.
[4] Matthew Fenwick, Jeffrey C. Hoch, Eldon Ulrich, and Michael R. Gryk. Connjur r: an annotation strategy for fostering reproducibility in bio-nmr---protein spectral assignment. Journal of Biomolecular NMR, 63(2):141--150, Oct 2015. [ DOI ]
Reproducibility is a cornerstone of the scientific method, essential for validation of results by independent laboratories and the sine qua non of scientific progress. A key step toward reproducibility of biomolecular NMR studies was the establishment of public data repositories (PDB and BMRB). Nevertheless, bio-NMR studies routinely fall short of the requirement for reproducibility that all the data needed to reproduce the results are published. A key limitation is that considerable metadata goes unpublished, notably manual interventions that are typically applied during the assignment of multidimensional NMR spectra. A general solution to this problem has been elusive, in part because of the wide range of approaches and software packages employed in the analysis of protein NMR spectra. Here we describe an approach for capturing missing metadata during the assignment of protein NMR spectra that can be generalized to arbitrary workflows, different software packages, other biomolecules, or other stages of data analysis in bio-NMR. We also present extensions to the NMR-STAR data dictionary that enable machine archival and retrieval of the “missing” metadata.
[3] Therese N. Tripler, Mark W. Maciejewski, Carolyn M. Teschke, and Andrei T. Alexandrescu. NMR assignments for the insertion domain of bacteriophage CUS-3 coat protein. Biomolecular NMR Assignments, 9(2):333--336, Feb 2015. [ DOI ]
CUS-3 is a P22-like tailed dsDNA bacteriophage that infects Escherichia coli serotype K1. The CUS-3 coat protein, which forms the icosahedral capsid, has a conserved HK97-fold but with a non-conserved accessory domain known as the insertion domain (I-domain). Sequence alignment of the coat proteins from CUS-3 and P22 shows higher sequence similarity for the I-domains (35 %) than for the HK97-cores, suggesting the I-domains play important functional roles. The I-domain of the P22 coat protein, which has an NMR structure comprised of a six-stranded β-barrel, has been shown to govern the assembly, stability and size of the resulting capsid particles. Here, we report the 1H, 15N, and 13C assignments for the I-domain from the coat protein of bacteriophage CUS-3. The secondary structure and dynamics of the CUS-3 I-domain, predicted from the assigned NMR chemical shifts, agree with those of the P22 I-domain, suggesting the CUS-3 and P22 I-domains may have similar structures and functions in capsid assembly.
[2] Rebecca L. Newcomer, LaTasha C.R. Fraser, Carolyn M. Teschke, and Andrei T. Alexandrescu. Mechanism of protein denaturation: Partial unfolding of the P22 coat protein I-Domain by urea binding. Biophysical Journal, 109(12):2666 -- 2677, 2015. [ DOI ]
The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC' H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility.
[1] Alessandro A. Rizzo, Margaret M. Suhanovsky, Matthew L. Baker, LaTasha C.R. Fraser, Lisa M. Jones, Don L. Rempel, Michael L. Gross, Wah Chiu, Andrei T. Alexandrescu, and Carolyn M. Teschke. Multiple functional roles of the accessory I-Domain of bacteriophage P22 coat protein revealed by NMR structure and CryoEM modeling. Structure, 22(6):830 -- 841, 2014. [ DOI ]
Some capsid proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 coat protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of coat protein. The I-domain has an antiparallel six-stranded β-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric coat protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 coat protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the coat protein HK97-core.

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