A5041238336- Electron Spin Resonance Studies
- Lanthanide and Transition Metal Complexes
- Advanced NMR Techniques and Applications
- Magnetism in coordination complexes
- Photosynthetic Processes and Mechanisms
- Advanced MRI Techniques and Applications
- Bacterial biofilms and quorum sensing
- Photoreceptor and optogenetics research
- Spectroscopy and Quantum Chemical Studies
- Advanced Fluorescence Microscopy Techniques
- Advanced Electron Microscopy Techniques and Applications
- Protein Structure and Dynamics
- Endoplasmic Reticulum Stress and Disease
- Streptococcal Infections and Treatments
- Mitochondrial Function and Pathology
- Antibiotic Resistance in Bacteria
- Plant Pathogenic Bacteria Studies
- Mass Spectrometry Techniques and Applications
- ATP Synthase and ATPases Research
- Orbital Angular Momentum in Optics
- Antimicrobial Resistance in Staphylococcus
- Force Microscopy Techniques and Applications
- Antimicrobial Peptides and Activities
- Infections and bacterial resistance
- Vibrio bacteria research studies
University of Washington
2020-2024
Medical College of Wisconsin
2018-2023
University of Wisconsin–Milwaukee
2013
In nature, proteins that switch between two conformations in response to environmental stimuli structurally transduce biochemical information a manner analogous how transistors control flow computing devices. Designing with distinct but fully structured is challenge for protein design as it requires sculpting an energy landscape minima. Here we describe the of “hinge” populate one designed state absence ligand and second presence ligand. X-ray crystallography, electron microscopy, double...
Here we introduce chiLife, a Python package for site-directed spin label (SDSL) modeling electron paramagnetic resonance (EPR) spectroscopy, in particular double electron–electron (DEER). It is based on silico attachment of rotamer ensemble representations labels to protein structures. chiLife enables the development custom analysis and pipelines using SDSL EPR experimental data. allows user add labels, scoring functions methods. designed with integration into third-party software mind, take...
Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a powerful technique for the investigation of protein structure and dynamics. Accurate spin-label modeling methods are essential to make full quantitative use site-directed data model validation. Using set double electron-electron from seven different site pairs on maltodextrin/maltose-binding under two conditions using five spin labels, we compare ability widely used methods, based accessible volume sampling rotamer...
Dipolar electron paramagnetic resonance (EPR) experiments such as double electron-electron (DEER) measure distributions of nanometer-scale distances between unpaired electrons, which provide valuable information for structural characterization proteins and other macromolecular systems. To determine these from the experimental signal, it is critical to employ an accurate model signal. For dilute samples doubly spin-labeled molecules, signal a product intramolecular intermolecular...
The ExoU type III secretion enzyme is a potent phospholipase A2 secreted by the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. Activation of activity induced protein-protein interactions with ubiquitin in cytosol targeted eukaryotic cell, leading to destruction host cell membranes. Previous work our laboratory suggested that conformational changes within C-terminal domain toxin might be involved activation mechanism. In this study, we use site-directed spin-labeling electron...
Significance Structure–function aspects of dynamic, membrane-associated proteins are difficult to mechanistically define. In this study, computational and biophysical methods applied identify a ubiquitin-interaction domain essential for the activity bacterial cytotoxin, ExoU. Introduction mutations that diminish or improve ubiquitin interaction alters biochemical biological activities toxin. This strategy these verification procedures may be useful where traditional NMR crystallographic...
Achromobacter xylosoxidans is increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that A. plays in pathology remains unknown. This knowledge gap largely due to lack model systems available study toxic potential this bacterium. Recently, phospholipase A 2 (PLA ) encoded by majority genomes, termed AxoU, was identified.
Protein design has focused primarily on the of ground states, ensuring they are sufficiently low energy to be highly populated. Designing kinetics and dynamics a system requires, in addition, excited states that traversed transitions from one low-lying state another. This is challenging task as such must strained poorly populated, but not so populated at all, because protein methods have generally creating near-ideal structures. Here we describe general approach for designing systems which...
Abstract ExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa , serves as prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used identify an unstructured loop approximately 20 residues in amino acid sequence. Mutational analyses indicate importance specific mediating catalytic activity. Engineered disulfide...
ExoU is a 74 kDa cytotoxin that undergoes substantial conformational changes as part of its function, is, it has multiple thermodynamically stable conformations interchange depending on environment. Such flexible proteins pose unique challenges to structural biology: (1) not only often difficult determine structures by X-ray crystallography for all biologically relevant because the flat energy landscape (2) but also experimental conditions can easily perturb conformation. The first challenge...
Bacterial phospholipases act as intracellular and extracellular enzymes promoting the destruction of phospholipid barriers inflammation during infections. Identifying with a common mechanism activation is an initial step in understanding structural functional properties. These properties serve critical information for design specific inhibitors to reduce enzymatic activity ameliorate host cell death. In this study, we identify verify cytotoxic PLA 2 from several bacterial pathogens. Similar...
The bifunctional spin label double-histidine copper-(II) capped with nitrilotriacetate [dHis-Cu(II)-NTA], used in conjunction electron paramagnetic resonance (EPR) methods can provide high-resolution distance data for investigating protein structure and backbone conformational diversity. Quantitative utilization of this is limited due to a lack rapid accurate dHis-Cu(II)-NTA modeling that be translate experimental into restraints. Here, we develop two rotamer libraries using set recently...
ABSTRACT With the great progress on determining protein structures over last decade comes a renewed appreciation that must be combined with dynamics and energetics to understand function. Fluorescence spectroscopy, specifically Förster resonance energy transfer (FRET), provides window into due its application at physiological temperatures ability measure ångström scale. We have recently advanced transition metal FRET (tmFRET) study allosteric regulation of maltose binding reported...
Abstract Proteins that switch between two structural states as a function of environmental stimuli are widespread in nature. These proteins structurally transduce biochemical information manner analogous to how transistors control flow computing devices. Engineering challenges ranging from biological devices molecular motors require such two-state switches, but designing these is an unsolved problem it requires sculpting energy landscape with low-energy distinct conformations can be...
Proteins are the workhorses of biology, orchestrating a myriad cellular functions through intricate conformational changes. Protein allostery, phenomenon where binding ligands or environmental changes induce rearrangements in protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used interrogate associated with protein allostery and recently introduced novel FRET acceptors utilizing metal-bipyridyl...
Abstract Here we introduce chiLife, a Python package for site-directed spin label (SDSL) modeling electron paramagnetic resonance (EPR) spectroscopy, in particular double electron–electron (DEER). It is based on silico attachment of rotamer ensemble representations labels to protein structures. chiLife enables the development custom analysis and pipelines using SDSL EPR experimental data. allows user add labels, scoring functions methods. designed with integration into third-party software...