John L. Markley

• Ph.D.
• University of Wisconsin–Madison

Background: Niacin-derived nicotinamide adenine dinucleotide is an essential cofactor for many dehydrogenase enzymes involved in vitamin A (VA) metabolism. Several countries with high prevalence of VA deficiency rely on maize, a poor source of available niacin, as a dietary staple. Objective: This study evaluated the interaction of dietary niaci...

The heightened dipolar interactions in solids render solid-state NMR (ssNMR) spectra more difficult to interpret than solution NMR spectra. On the other hand, ssNMR does not suffer from severe molecular weight limitations like solution NMR. In recent years, ssNMR has undergone rapid technological developments that have enabled structure–function st...

The heightened dipolar interactions in solids render solid-state NMR (ssNMR) spectra more difficult to interpret than solution NMR spectra. On the other hand, ssNMR does not suffer from severe molecular weight limitations like solution NMR. In recent years, ssNMR has undergone rapid technological developments that have enabled structure–function st...

NMR spectroscopy provides structural and functional information about biomolecules and their complexes. The complexity of these systems can make the NMR data difficult to interpret, particularly for newer users of NMR technology, who may have limited understanding of the tools available and how they are used. To alleviate this problem, we have crea...

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four α-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized histone...

The ATPase Family, AAA domain-containing protein 2 (ATAD2) bromodomain (BRD) has a canonical bromodomain structure consisting of four alpha-helices. ATAD2 functions as a co-activator of the androgen and estrogen receptors as well as the MYC and E2F transcription factors. ATAD2 also functions during DNA replication, recognizing newly synthesized his...

Protein Data Bank is the single worldwide archive of experimentally determined macromolecular structure data. Established in 1971 as the first open access data resource in biology, the PDB archive is managed by the worldwide Protein Data Bank (wwPDB) consortium which has four partners—the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Dat...

Peak picking is a critical step in biomolecular NMR spectroscopy. The program, iPick, presented here provides a scripting tool and a graphical user interface (GUI), which allow the user to perform interactive and intuitive peak picking and validation. The click-and-run GUI requires no computer programming skills, while the scripting tool can be use...

Bromodomains exhibit preferences for specific patterns of post-translational modifications on core and variant histone proteins. We examined the ligand specificity of the ATAD2B bromodomain and compared it to its closely related paralogue in ATAD2. We show that the ATAD2B bromodomain recognizes mono- and diacetyllysine modifications on histones H4...

Motivation: Correlated NMR chemical shift changes identified through the CHEmical Shift Projection Analysis (CHESPA) and CHEmical Shift Covariance Analysis (CHESCA) reveal pathways of allosteric transitions in biological macromolecules. To address the need for an automated platform that implements CHESPA and CHESCA and integrates them with other N...

Significance The proto-oncogene KRAS , a small GTPase, is frequently mutated in pancreatic, colorectal, and lung cancer. These mutations result in elevated levels of the activated guanosine triphosphate-bound form of KRAS. Localized at the plasma membrane, KRAS functions to recruit effectors, predominantly RAF kinase for activation and initiation o...

The chemical composition of saccharide complexes underlies their biomedical activities as biomarkers for cardiometabolic disease, various types of cancer, and other conditions. However, because these molecules may undergo major structural modifications, distinguishing between compounds of saccharide and non-saccharide origin becomes a challenging c...

Bromodomains are chromatin reader modules that recognize acetylated lysine. Different bromodomains exhibit a preference for specific patterns of lysine acetylation marks on core and variant histone proteins, however, the functional relationships that exist between histone acetyllysine ligands and bromodomain recognition remain poorly understood. In...

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex...

The Ebola Virus is a causative agent of viral hemorrhagic fever outbreaks and a potential global health risk. The outbreak in West Africa (2013-2016) led to 11,000+ deaths and 30,000+ Ebola infected individuals. The current outbreak in the Democratic Republic of Congo (DRC) with 3,000+ confirmed cases and 2,000+ deaths attributed to Ebola virus inf...

New drugs are needed for glioblastoma, an aggressive brain tumor with a dismal prognosis. We recently reported that gallium maltolate (GaM) retards the growth of glioblastoma in a rat orthotopic brain tumor model by inhibiting mitochondrial function and iron-dependent ribonucleotide reductase (RR). However, GaM's mechanism of action at the mitochon...

NFU1 is a late acting factor in the biogenesis of human mitochondrial iron-sulfur proteins. Mutations in NFU1 are associated with genetic diseases such as multiple mitochondrial dysfunctions syndrome 1 (MMDS1) that involve defects in mitochondrial [4Fe-4S] proteins. We present results from NMR spectroscopy, small angle X-ray scattering, size exclus...

Synaptosomes are isolated nerve terminals that contain synaptic components, including neurotransmitters, metabolites, adhesion/fusion proteins, and nerve terminal receptors. The essential role of synaptosomes in neurotransmission has stimulated keen interest in understanding both their proteomic and metabolic composition. Mass spectrometric (MS) qu...

Bromodomain-containing proteins are often part of chromatin-modifying complexes, and their activity can lead to altered expression of genes that drive cancer, inflammation and neurological disorders in humans. Bromodomain-PHD finger protein 1 (BRPF1) is part of the MOZ (monocytic leukemic zinc-finger protein) HAT (histone acetyltransferase) complex...

The Biological Magnetic Resonance Data Bank (BioMagResBank or BMRB), founded in 1988, serves as the archive for data generated by nuclear magnetic resonance (NMR) spectroscopy of biological systems. NMR spectroscopy is unique among biophysical approaches in its ability to provide a broad range of atomic and higher-level information relevant to the...

Motivation: Two-dimensional [15N-1H] separated local field (SLF) solid-state NMR experiments of membrane proteins aligned in lipid bilayers provide tilt and rotation angles for α-helical segments using Polar Index Slant Angle (PISA) wheel models. No integrated software has been made available for data analysis and visualization. Results: We have...

The self-assembly of collagen-mimetic peptides (CMPs) that form sticky-ended triple helices has allowed the production of surprisingly stable artificial collagen fibers and hydrogels. Assembly through sticky ends requires the recognition of a single strand by a templated strand dimer. Although CMPs and their triple helices have been studied extensi...

We describe an approach to improving the quality and interoperability of open data related to small molecules, such as metabolites, drugs, natural products, food additives, and environmental contaminants. The approach involves computer implementation of an extended version of the IUPAC International Chemical Identifier (InChI) system that utilizes...

Human myeloid-derived growth factor (hMYDGF) is a 142-residue protein with a C-terminal endoplasmic reticulum (ER) retention sequence (ERS). Extracellular MYDGF mediates cardiac repair in mice after anoxic injury. Although homologs of hMYDGF are found in eukaryotes as distant as protozoans, its structure and function are unknown. Here we present th...

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss furth...

Ebola viral infections have resulted in several deadly epidemics in recent years in West and Central Africa. Because only one of the seven proteins encoded by the viral genome possesses enzymatic activity, disruption of protein–protein interactions is a promising route for antiviral drug development. We carried out a screening campaign to identify...

Metabolomics is the study of profiles of small molecules in biological fluids, cells, or organs. These profiles can be thought of as the “fingerprints” left behind from chemical processes occurring in biological systems. Because of its potential for groundbreaking applications in disease diagnostics, biomarker discovery, and systems biology, metabo...

The mechanisms by which cells adapt to proteotoxic stress are largely unknown, but are key to understanding how tumor cells, particularly in vivo, are largely resistant to proteasome inhibitors. Analysis of cancer cell lines, mouse xenografts and patient-derived tumor samples all showed an association between mitochondrial metabolism and proteasome...

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Various methods for understanding the structural and dynamic properties of proteins rely on the analysis of their NMR chemical shifts. These methods require the initial assignment of NMR signals to particular atoms in the sequence of the protein, a step that can be very time-consuming. The probabilistic interaction network of evidence (PINE) algori...

The J-domain protein Zuotin is a multi-domain eukaryotic Hsp70 co-chaperone. Though it is primarily ribosome-associated, positioned at the exit of the 60S subunit tunnel where it promotes folding of nascent polypeptide chains, Zuotin also has off-ribosome functions. Domains of Zuotin needed for 60S association and interaction with Hsp70 are conserv...

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine‐rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well‐resolved nuclear magnetic resonance (NMR) spectra and also...

This letter announces that PDBx/mmCIF format files will become mandatory for crystallographic depositions to the Protein Data Bank (PDB).

Identification of discrepant data in aggregated databases is a key step in data curation and remediation. We have applied the ALATIS approach, which is based on the international chemical shift identifier (InChI) model, to the full PubChem Compound database to generate unique and reproducible compound and atom identifiers for all entries for which...

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...

Caloric Restriction (CR) without malnutrition delays aging and the incidence of age‐related diseases. Adult‐onset CR enhances survival and reduces age‐related mortality in rhesus monkey ( Macaca mulatta ) compared to ad libitum fed controls, suggesting that the longevity benefits of CR can apply to primates, including humans; however, the mechanism...

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....

Iron-sulfur (Fe-S) clusters, the ubiquitous protein cofactors found in all kingdoms of life, perform a myriad of functions including nitrogen fixation, ribosome assembly, DNA repair, mitochondrial respiration, and metabolite catabolism. The biogenesis of Fe-S clusters is a multi-step process that involves the participation of many protein partners....

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 a...

Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we...

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...

The sections in this article are Introduction Project Data Handling: S esame ORF Cloning E . c oli Cell‐Based Protein Production Pipeline Wheat Germ Cell‐Free Protein Production Mass Spectrometry of Purified Proteins for Quality Assurance and Analysis Crystallomics and X ‐ R ay Structure Determination NMR Screening and Structure Determinati...

The mechanisms used by cancer cells to resist the severe disruption in protein homeostasis caused by proteasome inhibitors remain obscure. Here, we show this resistance correlates with a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Employing small molecule screens, we identified a striking overlap between compounds that pr...

Caloric restriction (CR) extends lifespan and delays the onset of age-related disorders in diverse species. Metabolic regulatory pathways have been implicated in the mechanisms of CR, but the molecular details have not been elucidated. Here, we show that CR engages RNA processing of genes associated with a highly integrated reprogramming of hepatic...

Frataxin (FXN) is involved in mitochondrial iron‑sulfur (Fe-S) cluster biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency is associated with Friedreich ataxia, a neurodegenerative disease. We have used a combination of isothermal titration calorimetry and multinuclear NMR spectroscopy to investigate interactions among the co...

The Protein Data Bank (PDB) is the single global repository for experimentally determined 3D structures of biological macromolecules and their complexes with ligands. The worldwide PDB (wwPDB) is the international collaboration that manages the PDB archive according to the FAIR principles: Findability, Accessibility, Interoperability and Reusabilit...

Whereas iron-sulfur (Fe-S) cluster assembly on the wild-type scaffold protein ISCU, as catalyzed by the human cysteine desulfurase complex (SDA), exhibits a requirement for frataxin (FXN), assembly on variant ISCU(M108I) has been shown to bypass the FXN requirement. Wild-type ISCU populates two interconverting conformational states: one structured...

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 st...

Nuclear magnetic resonance (NMR) spectroscopy is one of the three major approaches for determining the structures of biological macromolecules. Historically, NMR spectroscopy was number two after X-ray crystallography in the rate of depositions to the Protein Data Bank (PDB). However, electron cryomicroscopy (CryoEM) recently surpassed NMR in this...

NMR (nuclear magnetic resonance) spectroscopy, along with X-ray crystallography and Cryo-EM, 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...

Endogenous production of α-dicarbonyls by lactic acid bacteria can influence the quality and consistency of fermented foods and beverages. Methylglyoxal (MG) in Parmesan cheese can contribute toward undesired browning during low temperature ripening and storage conditions, leading to the economic depreciation of affected cheeses. We demonstrate the...

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 pola...

The Worldwide PDB recently launched a deposition, biocuration, and validation tool: OneDep. At various stages of OneDep data processing, validation reports for three-dimensional structures of biological macromolecules are produced. These reports are based on recommendations of expert task forces representing crystallography, nuclear magnetic resona...

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small mole...

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the fun...

NMR and refinement statistics for Ydj1 J-domain structure. Statistics from the validation reports for the NMR structures of Ydj1 determined by PONDEROSA-C/S with final step calculation with subsequent explicit water refinement option that selects best 20 of 100 structures based on lowest pseudo-potential energy criteria. PROCHECK and MOLPROBITY val...

Interaction of Ssa1 (WT and variants) with peptide P5. Equilibrium binding of F-P5 over a range of Ssa1 concentrations. Indicated concentrations of Ssa1 were combined with F-P5 (10 nM final concentration) in the presence of 1 mM ADP. Reactions were incubated at room temperature until equilibrium was reached. Symbols represent the mean of 4 independ...

Analysis of prion aggregates in [PSI+]Sc37 and [RNQ+] sis1-Δ strains carrying ydj1 or ssa1 mutations. (A) Cell lysates, made from WY26 strains in which the URA3 marked plasmid carrying WT SIS1 was shuffled with plasmids carrying the indicated ydj1 or ssa1 mutant gene, were separated using SDD-AGE, transferred to nitrocellulose and immunoblotted wit...

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...

Burley et al. (leadership of the Worldwide PDB [wwPDB] Partnership [wwpdb.org] and the wwPDB Integrative/Hybrid Methods Task Force) announce public release of a prototype system for depositing integrative/hybrid structural models, PDB-Development (PDB-Dev; https://pdb-dev.wwpdb.org).

A polyether antibiotic, ecteinamycin (1) was isolated from a marine Actinomadura sp., cultivated from the ascidian Ectein-ascidia turbinata. 13C-enrichment, high resolution NMR spectroscopy and molecular modeling enabled elucidation of the structure of 1 which was validated on the basis of comparisons with its recently reported crystal structure. I...

Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of internuclear vectors. We adapted a force field structure calculation methodology to allow free sampling of both R and...

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small mole...

The Protein Data Bank (PDB)––the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes––was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Prote...

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...

Escherichia coli has been the most widely used host to produce large amounts of heterologous proteins. However, given an input plasmid DNA, E. coli may produce soluble protein, produce only inclusion bodies, or yield little or no protein at all. Many efforts have been made to surmount these problems, but most of them have involved time-consuming an...

A list of genes studied in this work. (XLSX)

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...

Mitochondrial cysteine desulfurase is an essential component of the machinery for iron-sulfur cluster biosynthesis. Human cysteine desulfurase that is catalytically active in vitro can be prepared by overexpressing two protein components of this system, ISD11 and NFS1, in Escherichia coli cells. We report that ISD11 and NFS1 form a complex containi...

OneDep, a unified system for deposition, biocuration, and validation of experimentally determined structures of biological macromolecules to the PDB archive, has been developed as a global collaboration by the worldwide PDB (wwPDB) partners. This new system was designed to ensure that the wwPDB could meet the evolving archiving requirements of the...

Multi-nuclear, multi-dimensional Fourier transform (FT) NMR spectroscopy has ushered in a broad range of new applications in chemistry and biology. Multi-dimensional NMR experimental data comes at a price, however. Additional dimensions lead to exponential increases in data collection time, and the number of data points required increases at polyno...

Ferredoxins play an important role as an electron donor in iron-sulfur (Fe-S) cluster biosynthesis. Two ferredoxins, FDX1 and FDX2, are present in the matrix of human mitochondria. Conflicting results have been reported regarding their respective function in mitochondrial iron-sulfur cluster biogenesis. We report here biophysical studies of the int...

Human mitochondrial NFU1 functions in the maturation of iron-sulfur proteins, and NFU1 deficiency is associated with a fatal mitochondrial disease. We determined three-dimensional structures of the N- and C-terminal domains of human NFU1 by nuclear magnetic resonance spectroscopy and used these structures along with small-angle X-ray scattering (SA...

The global Protein Data Bank (PDB) was the first open-access digital archive in biology. The history and evolution of the PDB are described, together with the ways in which molecular structural biology data and information are collected, curated, validated, archived, and disseminated by the members of the Worldwide Protein Data Bank organization (w...

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 a...

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 dyn...

CCollisions between DNA replication complexes (replisomes) and barriers such as damaged DNA or tightly bound protein complexes can dissociate replisomes from chromosomes prematurely. Replisomes must be reloaded under these circumstances to avoid incomplete replication and cell death. Bacteria have evolved multiple pathways that initiate DNA replica...

Protein misfolding and aggregation are pathological events that place a significant amount of stress on the maintenance of protein homeostasis (proteostasis). For prevention and repair of protein misfolding and aggregation, cells are equipped with robust mechanisms that mainly rely on molecular chaperones. Two classes of molecular chaperones, heat...