A5017903418- Bacterial Genetics and Biotechnology
- Enzyme Structure and Function
- Protein Structure and Dynamics
- Drug Transport and Resistance Mechanisms
- RNA and protein synthesis mechanisms
- Bacteriophages and microbial interactions
- Legume Nitrogen Fixing Symbiosis
- Lipid Membrane Structure and Behavior
- Escherichia coli research studies
- Biochemical and Molecular Research
- Antibiotic Resistance in Bacteria
- Plant Micronutrient Interactions and Effects
- Viral Infectious Diseases and Gene Expression in Insects
- Photosynthetic Processes and Mechanisms
- Potassium and Related Disorders
- Metalloenzymes and iron-sulfur proteins
- DNA and Nucleic Acid Chemistry
- RNA Interference and Gene Delivery
- Mass Spectrometry Techniques and Applications
- Plant Pathogenic Bacteria Studies
- Electron Spin Resonance Studies
- Plant-based Medicinal Research
- Chemical Synthesis and Analysis
- Porphyrin Metabolism and Disorders
- Metal-Catalyzed Oxygenation Mechanisms
Universität Hamburg
2016-2024
Hamburg Centre for Ultrafast Imaging
2019
Max Planck Institute for the Structure and Dynamics of Matter
2018-2019
Institute of Biochemistry
2019
Center for Free-Electron Laser Science
2018
University of Glasgow
2014-2016
Abstract Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of method to study structure an IMP, solubilization from usually first step workflow. Here, we establish a simple, high-throughput screening identify optimal conditions protein stabilization. We apply differential scanning fluorimetry combination with scattering upon thermal denaturation unfolding proteins. Nine different prokaryotic...
Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, novel member this family from Pseudomonas aeruginosa, targets susceptible strains species through recognition the common polysaccharide antigen (CPA) P. aeruginosa lipopolysaccharide is predominantly homopolymer d-rhamnose. Structural biophysical analyses CPA occurs C-terminal carbohydrate-binding domain L1 interaction prerequisite for...
Many microbes and fungi acquire the essential ion Fe3+ through synthesis secretion of high-affinity chelators termed siderophores. In Gram-negative bacteria, these ferric-siderophore complexes are actively taken up using highly specific TonB-dependent transporters (TBDTs) located in outer bacterial membrane (OM). However, detailed mechanism how inner-membrane protein TonB connects to OM as well interplay between siderophore- TonB-binding transporter is still poorly understood. Here, we...
Critical assessment of structure prediction (CASP) conducts community experiments to determine the state art in computing protein from amino acid sequence. The process relies on experimental providing information about not yet public or be solved structures, for use as targets. For some targets, is time CASP. Calculated accuracy improved dramatically this round, implying that models should now much more useful resolving many sorts difficulties. To test this, selected seven unsolved targets...
Increasing rates of antibiotic resistance among Gram-negative pathogens such as Pseudomonas aeruginosa means alternative approaches to development are urgently required. Pyocins, produced by P. for intraspecies competition, highly potent protein antibiotics known actively translocate across the outer membrane aeruginosa. Understanding and exploiting mechanisms which pyocins target, penetrate kill is a promising approach development. In this work we show therapeutic potential newly identified...
Attaching and effacing pathogens overcome colonisation resistance by competing with metabolically similar organisms for limited resources. Enterohaemorrhagic E. coli (EHEC) utilises the pathogenicity island-encoded Accessory ʟ-arabinose Uptake (Aau) transporter to effectively colonise mouse gut, hypothesised be achieved via an enhanced capacity scavenge ʟ-arabinose. Aau is regulated exclusively in response ʟ-arabinose, but it unclear how this system specifically benefits EHEC vivo. Here, we...
Serial synchrotron crystallography allows low X-ray dose, room-temperature crystal structures of proteins to be determined from a population microcrystals. Protein production and crystallization is non-trivial procedure it essential have X-ray-compatible sample environments that keep consumption the crystals in their native environment. This article presents fast optimized manufacturing route metal-polyimide microfluidic flow-focusing devices which allow for collection diffraction data flow....
Recent structures of full-length ATP-binding cassette (ABC) transporter MsbA in different states indicate large conformational changes during the reaction cycle that involve transient dimerization its nucleotide-binding domains (NBDs). However, a detailed molecular understanding structural and associated kinetics upon ATP binding hydrolysis is still missing. Here, we employed time-resolved small-angle X-ray scattering, initiated by stopped-flow mixing, to investigate accompanying NBD (upon...
Iron is a key nutrient for almost all living organisms. Paradoxically, it poorly soluble and consequently bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists use siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able produce their own siderophores or those produced by other microorganisms (exosiderophores) in piracy strategy. Pseudomonas aeruginosa produces two pyoverdine...
Gram-negative bacteria take up the essential ion Fe3+ as ferric-siderophore complexes through their outer membrane using TonB-dependent transporters. However, subsequent route inner differs across many bacterial species and siderophore chemistries is not understood in detail. Here, we report crystal structure of protein FoxB (from Pseudomonas aeruginosa) that involved Fe-siderophore uptake. The revealed a fold with two tightly bound heme molecules. In combination vitro reduction assays vivo...
Plasma-membrane Ca2+-ATPases expel Ca2+ from the cytoplasm and are key regulators of homeostasis in eukaryotes. They autoinhibited under low concentrations. Calmodulin (CaM)-binding to a unique regulatory domain releases autoinhibition activates pump. However, structural basis for this activation, including overall structure calcium pump its complex with calmodulin, is unknown. We previously determined high-resolution calmodulin plasma-membrane Ca2+-ATPase ACA8 revealed bimodular mechanism...
This work demonstrates a new method for investigating time-resolved structural changes in protein conformation and oligomerization via photocage-initiated X-ray solution scattering by observing the ATP-driven dimerization of MsbA nucleotide-binding domain. Photocaged small molecules allow observation single-turnover reactions non-naturally photoactivatable proteins. The kinetics reaction can be derived from associated with ATP-binding subsequent dimerization. expanded to any...
Abstract Iron is a limiting nutrient in bacterial infection putting it at the centre of an evolutionary arms race between host and pathogen. Gram-negative bacteria utilize TonB-dependent outer membrane receptors to obtain iron during infection. These acquire either concert with soluble iron-scavenging siderophores or through direct interaction extraction from proteins. Characterization these provides invaluable insight into pathogenesis. However, only subset virulence-related have been...
The outer membrane of gram-negative bacteria prevents many antibiotics from reaching intracellular targets. However, some antimicrobials can take advantage iron import transporters to cross this barrier. We showed previously that the thiopeptide antibiotic thiocillin exploits nocardamine xenosiderophore transporter, FoxA, opportunistic pathogen Pseudomonas aeruginosa for uptake. Here, we show FoxA also transports bisucaberin and describe at 2.5 Å resolution crystal structure bound FoxA....
The ATP‐binding cassette transporter MsbA is a lipid flippase, translocating A, glycolipids, and lipopolysaccharides from the inner to outer leaflet of membrane Gram‐negative bacteria. It has been used as model system for time‐resolved structural studies several structures in different states reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due limited resolution available structures, detailed information on bound nucleotides remained elusive. Here, we have...
Summary The colicin‐like bacteriocins are potent protein antibiotics that have evolved to efficiently cross the outer membrane of Gram‐negative bacteria by parasitizing nutrient uptake systems. We structurally characterized colicin M ‐like bacteriocin, pectocin 2, which is active against strains P ectobacterium spp. This unusual bacteriocin lacks intrinsically unstructured translocation domain usually mediates these across membrane, containing only a single globular ferredoxin connected its...
How ultra-high-affinity protein–protein interactions retain high specificity is still poorly understood. The interaction between colicin DNase domains and their inhibitory immunity (Im) proteins an that essential for the neutralisation of endogenous catalytic activity protection against exogenous bacteriocins. DNase–Im a model system study high-affinity interactions. However, despite fact closely related colicin-like bacteriocins are widely produced by Gram-negative bacteria, this has only...
Calmodulin (CaM) is a very conserved, ubiquitous, eukaryotic protein that binds four Ca2+ ions with high affinity. It acts as calcium sensor by translating signals into cellular processes such metabolism, inflammation, immune response, memory, and muscle contraction. Calcium binding to CaM leads conformational changes enable Ca2+/CaM recognize bind various target proteins The mode partners of are diverse, consensus sequence lacking. Here, we describe an elegant system allows...
Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding the mechanism import such potent has increased significantly in recent years, especially with reporting several structures bacteriocin domains. Less well understood is structural biochemistry intact bacteriocins how these compare across bacterial species. Here, we focus on endonuclease (DNase) target...
Membrane protein research suffers from the drawback that detergents, which are commonly used to solubilize integral membrane proteins (IMPs), often lead instability and reduced activity. Recently, lipid nanodiscs (NDs) saposin-lipoprotein particles (Salipro) have emerged as alternative carrier systems keep in a native-like lipidic solution environment suitable for biophysical structural studies. Here, we systematically compare Salipros with respect long-term stability well activity of...
A crucial bottleneck in membrane protein structural biology is the difficulty identifying a detergent that can maintain stability and functionality of integral proteins (IMPs). Detergents are poor mimics, their common use crystallography may be one reason for challenges obtaining high-resolution crystal structures many IMP families. Lipid-like peptides (LLPs) have detergent-like properties been proposed as alternatives solubilization G protein-coupled receptors other proteins. Here, we...
Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, X-ray structure protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism activation and conformational change essential for protease inhibition. In this competitive mechanism, cleavage bait-region domain results untethering an intrinsically disordered region disrupts native interdomain...