WILLIAM WESTLER - Academia.edu (original) (raw)

Papers by WILLIAM WESTLER

Antiviral Research, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Journal of Biomolecular NMR, 2019

Various methods for understanding the structural and dynamic properties of proteins rely on the a... more 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 PINE (Probabilistic Interaction Network of Evidence) algorithm for automated assignment of backbone and side chain chemical shifts utilizes a Bayesian probabilistic network model that analyzes sequence data and peak lists from multiple NMR experiments. PINE, which is one of the most popular and reliable automated chemical shift assignment algorithms, has been available to the protein NMR community for longer than a decade. We announce here a new web server version of PINE, called I-PINE (Integrative PINE), which supports more types of NMR experiments than PINE (including three-dimensional nuclear Overhauser enhancement (NOE) and four-dimensional J-coupling experiments) along with more comprehensive visualization of chemical shift based analysis of protein structure and dynamics.

Analytical chemistry, Jan 23, 2017

The exceptionally rich information content of nuclear magnetic resonance (NMR) spectra is routine... more 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 parameterizes 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 b...

Archives of biochemistry and biophysics, Aug 8, 2017

The editors of this special volume suggested this topic, presumably because of the perspective le... more The editors of this special volume suggested this topic, presumably because of the perspective lent by our combined >90-year association with biomolecular NMR. What follows is our personal experience with the evolution of the field, which we hope will illustrate the trajectory of change over the years. As for the future, one can confidently predict that it will involve unexpected advances. Our narrative is colored by our experience in using the NMR Facility for Biomedical Studies at Carnegie-Mellon University (Pittsburgh) and in developing similar facilities at Purdue (1977-1984) and the University of Wisconsin-Madison (1984-). We have enjoyed developing NMR technology and making it available to collaborators and users of these facilities. Our group's association with the Biological Magnetic Resonance data Bank (BMRB) and with the Worldwide Protein Data Bank (wwPDB) has also been rewarding. Of course, many groups contributed to the early growth and development of biomolecular...

Journal of biomolecular NMR, Apr 29, 2016

NMR spectroscopy is a powerful technique for determining structural and functional features of bi... more NMR spectroscopy is a powerful technique for determining structural and functional features of biomolecules in physiological solution as well as for observing their intermolecular interactions in real-time. However, complex steps associated with its practice have made the approach daunting for non-specialists. We introduce an NMR platform that makes biomolecular NMR spectroscopy much more accessible by integrating tools, databases, web services, and video tutorials that can be launched by simple installation of NMRFAM software packages or using a cross-platform virtual machine that can be run on any standard laptop or desktop computer. The software package can be downloaded freely from the NMRFAM software download page ( http://pine.nmrfam.wisc.edu/download_packages.html ), and detailed instructions are available from the Integrative NMR Video Tutorial page ( http://pine.nmrfam.wisc.edu/integrative.html ).

Journal of proteome research, Jan 11, 2016

NMR ligand affinity screening is a powerful technique that is routinely used in drug discovery or... more NMR ligand affinity screening is a powerful technique that is routinely used in drug discovery or functional genomics to directly detect protein-ligand binding events. Binding events can be identified by monitoring differences in the one-dimensional (1)H NMR spectrum of a compound with and without protein. Although a single NMR spectrum can be collected within a short period (2-10 min per sample), one-by-one screening of a protein against a library of hundreds or thousands of compounds requires a large amount of spectrometer time and a large quantity of protein. To improve the efficiency of these screens in both time and material, compounds are usually evaluated in mixtures ranging in size from 3 to 20 compounds. Ideally, the NMR signals from individual compounds in the mixture should not overlap so that spectral changes can be associated with a particular compound. We have developed a software tool, NMRmix, to assist in creating ideal mixtures from a large panel of compounds with k...

Journal of Biomolecular NMR, 2016

Data validation plays an important role in ensuring the reliability and reproducibility of studie... more Data validation plays an important role in ensuring the reliability and reproducibility of studies. NMR investigations of the functional properties, dynamics, chemical kinetics, and structures of proteins depend critically on the correctness of chemical shift assignments. We present a novel probabilistic method named ARECA for validating chemical shift assignments that relies on the nuclear Overhauser effect (NOE) data. ARECA has been evaluated through its application to 26 case studies and has been shown to be complementary to, and usually more reliable than, approaches based on chemical shift databases. ARECA is available online at http:// areca.nmrfam.wisc.edu/.

PLOS ONE, 2015

GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfe... more GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1 H-15 N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobY G153D , whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo-state and in complex with GTP. The CobY G153D :GTP complex was also found to be unstable over time.

Biophysical journal, 2015

IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, trave... more IscU, the scaffold protein for iron-sulfur (Fe-S) cluster biosynthesis in Escherichia coli, traverses a complex energy landscape during Fe-S cluster synthesis and transfer. Our previous studies showed that IscU populates two interconverting conformational states: one structured (S) and one largely disordered (D). Both states appear to be functionally important because proteins involved in the assembly or transfer of Fe-S clusters have been shown to interact preferentially with either the S or D state of IscU. To characterize the complex structure-energy landscape of IscU, we employed NMR spectroscopy, small-angle x-ray scattering (SAXS), and differential scanning calorimetry. Results obtained for IscU at pH 8.0 show that its S state is maximally populated at 25°C and that heating or cooling converts the protein toward the D state. Results from NMR and DSC indicate that both the heat- and cold-induced S→D transitions are cooperative and two-state. Low-resolution structural informatio...

Journal of Biomolecular NMR, 2015

The computationally demanding nature of automated NMR structure determination necessitates a deli... more The computationally demanding nature of automated NMR structure determination necessitates a delicate balancing of factors that include the time complexity of data collection, the computational complexity of chemical shift assignments, and selection of proper optimization steps. During the past two decades the computational and algorithmic aspects of several discrete steps of the process have been addressed. Although no single comprehensive solution has emerged, the incorporation of a validation protocol has gained recognition as a necessary step for a robust automated approach. The need for validation becomes even more pronounced in cases of proteins with higher structural complexity, where potentially larger errors generated at each step can propagate and accumulate in the process of structure calculation, thereby significantly degrading the efficacy of any software framework. This paper introduces a complete framework for protein structure determination with NMR-from data acquisition to the structure determination. The aim is twofold: to simplify the structure determination process for non-NMR experts whenever feasible, while maintaining flexibility by providing a set of modules that validate each step, and to enable the assessment of error propagations. This framework, called NMRFAM-SDF (NMRFAM-Structure Determination Framework), and its various components are available for download from the NMRFAM website (http://nmrfam.wisc.edu/software.htm).

Bulletin of the Korean Chemical Society, 2011

The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquit... more The vitamin D receptor binding peptide, VDRBP, was overexpressed as a fused form with the ubiquitin molecule in Rosetta(DE3)pLysS, a protein production strain of Escherichia coli harboring an induction controller plasmid. The fusion protein was bound to the immobilized metal ions, and the denaturation and renaturation of the fusion protein were performed as a part of the purification procedure. After the elution of the fusion protein, the peptide hormone was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The purity of the resulting peptide fragment was checked by MALDI-TOF mass and NMR spectroscopy. The final yields of the target peptide were around 5 and 2 mg per liter of LB and minimal media, respectively. The recombinant expression and purification of this peptide will enable structural and functional studies using multidimensional NMR spectroscopy and X-ray crystallography.

Biomolecular NMR Assignments, 2014

We report here backbone 1 H and 15 N assignments for RNase A obtained by using ADAPT-NMR, a fully... more We report here backbone 1 H and 15 N assignments for RNase A obtained by using ADAPT-NMR, a fully-automated approach for combined data collection, spectral analysis and resonance assignment. ADAPT-NMR was able to assign 98% of the resonances with 93% agreement with traditional data collection and assignment. Further refinement of the automated results with ADAPT-NMR Enhancer led to complete (100%) assignments with 96% agreement with assignments by the traditional approach. Biological Context Bovine pancreatic ribonuclease (RNase A) is a 124-residue protein that has served as a model for much landmark work on protein structure and function. RNase A is the third enzyme whose structure was determined by x-ray crystallography and is now the subject of more than 30 PDB entries. In addition, RNase A played a crucial role in the early development of NMR spectroscopy, leading to the determination of its solution structure. (For a review, see: Raines, 1998.) RNase A catalyzes the cleavage of the P-O 5′ bond of RNA with a k cat /K M value that can exceed 10 9 M −1 s −1 and exhibits high thermostability (T m 62 °C). (For a review, see: Lomax et al., 2012.) The presumed biological function of RNase A is catalysis of the depolymerization of ingested RNA. Yet, high levels of RNase A and its human homologue (RNase 1) in many different tissues are consistent with additional roles. (Futami et al. 1997; Wheeler et al. 2012). Recently, homologs and variants of RNase A have shown potential as cancer chemotherapeutic agents (Lomax et al. 2012). Enabling RNase A to achieve its clinical potential is likely to require a thorough understanding of its interactions with cellular molecules, including cell-surface glycans and the cytosolic ribonuclease inhibitor protein. Structural and dynamic aspects of these interactions can be probed by NMR spectroscopy. While characterizing these interactions, we found that our 1 H-15 N backbone assignments did not match with those reported previously (Shimotakahara et al. 1997). In particular, assignments to several amide peaks in flexible loop regions varied, despite the use of identical buffer conditions. We used the ADAPT-NMR method (Bahrami et al. 2012

Angewandte Chemie, 2014

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was d... more Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrateassociated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of 13 C-13 C gCOSY and 13 C-15 N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct 13 C-15 N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity.

PLoS Pathogens, 2009

Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes... more Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a Pol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic a-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a Pol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5-to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

Proteins: Structure, Function, and Bioinformatics, 2005

Binding sites in the full-length, ligand-binding domain of rat vitamin D receptor (LBD-rVDR) for ... more Binding sites in the full-length, ligand-binding domain of rat vitamin D receptor (LBD-rVDR) for an active hormone derived from vitamin D (1␣,25-dihydroxyvitamin D 3) and three of its C-2 substituted analogs were compared by nuclear magnetic resonance (NMR) spectroscopy. Specific residue labeled with [UL]-15 N 2 Trp allowed assignment of the side-chain H ⑀1 and N ⑀1 resonances of the single tryptophan residue at position 282 in LBD-rVDR. Comparison of 1 H[ 15 N] Heteronuclear Single Quantum Correlation (HSQC) spectra of apo and holo LBD-rVDR revealed that the position of the Trp282 H ⑀1 and N ⑀1 signals are sensitive to the presence of the ligand in the receptor cavity. Binding of the ligands to LBD-rVDR results in a shift of both Trp H ⑀1 and N ⑀1 resonances to lower frequencies. The results indicate that the interaction between the ligands and Trp282 is not responsible for differences in calcemic activity observed in vitamin D analogs. Proteins 2005;61:461-467.

Protein Expression and Purification, 2012

Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and ph... more Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from E. coli inclusion bodies. The heterologously expressed protein constructs retain full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with, 13 C, and 15 N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed.

Proceedings of the National Academy of Sciences, 2005

The rubredoxin from Clostridium pasteurianum ( Cp Rd) provides an excellent system for investigat... more The rubredoxin from Clostridium pasteurianum ( Cp Rd) provides an excellent system for investigating how the protein sequence modulates the reduction potential of the active site in an iron–sulfur protein. 15 N NMR spectroscopy has allowed us to determine with unprecedented accuracy the strengths of all six key hydrogen bonds between protein backbone amides and the sulfur atoms of the four cysteine residues that ligate the iron in the oxidized (Fe III ) and reduced (Fe II ) forms of wild-type Cp Rd and nine mutants (V44G, V44A, V44I, V44L, V8G, V8A, V8I, V8L, and V8G/V44G). The length (or strength) of each hydrogen bond was inferred from the magnitude of electron spin delocalized across the hydrogen bond from the iron atom onto the nitrogen. The aggregate lengths of these six hydrogen bonds are shorter in both oxidation states in variants with higher reduction potential than in those with lower reduction potential. Differences in aggregate hydrogen bonding upon reduction correlate l...

Journal of the Brazilian Chemical Society, 1999

Neste trabalho foi feito o estudo de ligação de espermina (SPM), N 1 , N 12-bismetilespermina (BM... more Neste trabalho foi feito o estudo de ligação de espermina (SPM), N 1 , N 12-bismetilespermina (BMS) e N 1 , N 12-bisetilespermina (BES) ao tRNA fen usando RMN de 1 H a 750 MHz. As poliaminas foram enriquecidas com 13 C nos resíduos 5-CH2 e 8-CH2, sendo obtidos os picos cruzados por efeito de Overhauser nuclear entre as ressonâncias dos hidrogênios dos metilenos marcados com 13 C e vários hidrogênios de grupos imino de pares de bases do tRNA fen através de espectros 1D filtrados por 13 C. Foi encontrado que, enquanto SPM e BMS se ligam ao N(3)-H dos pares de bases T54-m 1 A58, U50-A64 e U52-A62, BES liga-se só nos pares T54-m 1 A58 e U50-A64. Esta regiosseletividade de ligação das três poliaminas ao tRNA foi correlacionada com seus efeitos biológicos no crescimento celular. Usando células de câncer de melanoma humano (MALME-3M), foi encontrado que SPM e BMS não tem efeito e é citostático, respectivamente, enquanto que BES é claramente citotóxica. Esta última poliamina também afeta o ciclo celular e, contrário ao comportamento de SPM e BMS, acarreta a uma clara parada do ciclo celular G1 /S. The binding of spermine (SPM), N 1 ,N 12-bismethylspermine (BMS) and N 1 ,N 12-bisethylspermine (BES) to tRNA Phe was studied using 1 H-NMR at 750 MHz. The polyamines were enriched in 13 C at the 5-CH2 and 8-CH2 residues and the nuclear Overhauser enhancement (NOE) cross peaks connecting the 1 H-NMR resonances of the 13 C-methylenes and several base paired imino protons of tRNA Phe were obtained using 1D 13 C-half filtered spectra. It was found that while SPM and BMS bind to the N(3)-H of base pairs T54-m 1 A58, U50-A64 and U52-A62, BES binds only to T54m 1 A58 and U50-A64. This regioselectivity in the binding of the three polyamines to tRNA was correlated with their biological effects on cell growth. Using human melanoma cancer cells (MALME-3M), we found that SPM and BMS were without effect and cytostatic, respectively, while BES was distinctly cytotoxic. The latter also affected cell cycling and, at variance with SPM and BMS, lead to a distinct G1/S cell cycle arrest.

Journal of the American Chemical Society, 2010

The Rieske protein component of the cytochrome bc complex contains a [2Fe-2S] cluster ligated by ... more The Rieske protein component of the cytochrome bc complex contains a [2Fe-2S] cluster ligated by two cysteines and two histidines. We report here the pK a values of each of the imidazole rings of the two ligating histidines (His134 and His154) in the oxidized and reduced states of the Rieske protein from Thermus thermophilus (TtRp) as determined by NMR spectroscopy. Knowledge of these pK a values is of critical interest because of their pertinence to the mechanism of electron and proton transfer in the bifurcated Q-cycle. Although we earlier had observed the pH dependence of a 15 N NMR signal from each of the two ligand histidines in oxidized TtRp (Lin, I.

Journal of the American Chemical Society, 2012

A natural bond orbital (NBO) analysis of unpaired electron spin density in metalloproteins is pre... more A natural bond orbital (NBO) analysis of unpaired electron spin density in metalloproteins is presented, which allows a fast and robust calculation of paramagnetic NMR parameters. Approximately 90% of the unpaired electron spin density occupies metal−ligand NBOs, allowing the majority of the density to be modeled by only a few NBOs that reflect the chemical bonding environment. We show that the paramagnetic relaxation rate of protons can be calculated accurately using only the metal−ligand NBOs and that these rates are in good agreement with corresponding rates measured experimentally. This holds, in particular, for protons of ligand residues where the point-dipole approximation breaks down. To describe the paramagnetic relaxation of heavy nuclei, also the electron spin density in the local orbitals must be taken into account. Geometric distance restraints for 15 N can be derived from the paramagnetic relaxation enhancement and the Fermi contact shift when local NBOs are included in the analysis. Thus, the NBO approach allows us to include experimental paramagnetic NMR parameters of 15 N nuclei as restraints in a structure optimization protocol. We performed a molecular dynamics simulation and structure determination of oxidized rubredoxin using the experimentally obtained paramagnetic NMR parameters of 15 N. The corresponding structures obtained are in good agreement with the crystal structure of rubredoxin. Thus, the NBO approach allows an accurate description of the geometric structure and the dynamics of metalloproteins, when NMR parameters are available of nuclei in the immediate vicinity of the metal-site.