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Liquid crystals (LCs) are emerging as novel platforms for chemical, physical, and biological sensing. They can be used to detect biological amphiphiles such as lipids, fatty acids, digestive surfactants, and bacterial endotoxins. However, designing LC-based sensors in a manner that preserves their sensitivity and responsiveness to these stimuli, and possibly improves biocompatibility, remains challenging. In this work, the stabilization of LC droplets by oleosins, plant-sourced and highly surface active proteins due to their extended amphipathic helix, is investigated. Purified oleosins, at sub-micromolar concentrations, are shown to readily stabilize nematic LC droplets without switching their alignment, allowing them to detect surfactants at micromolar concentrations. Direct evidence of localization of oleosins at the LC–water interface is provided with fluorescent labeling, and the stabilized droplets remain stable over months. Interestingly, chiral LC droplets readily switch in the presence of nanomolar oleosin concentrations, an unexpected behavior that is explained by accounting for the energy barriers required for switching the alignment between the two cases. This leads thus to a twofold conclusion: oleosin-stabilized nematic LC droplets present a biocompatible alternative for bioanalyte detection, while chiral LCs can be further investigated for use as highly sensitive sensors for detecting amphipathic helices in biological systems.

Endomembrane remodeling to form a viral replication complex (VRC) is crucial for a virus to establish infection in a host. Although the composition and function of VRCs have been intensively studied, host factors involved in the assembly of VRCs for plant RNA viruses have not been fully explored. TurboID-based proximity labeling (PL) has emerged as a robust tool for probing molecular interactions in planta. However, few studies have employed the TurboID-based PL technique for investigating plant virus replication. Here, we used Beet black scorch virus (BBSV), an endoplasmic reticulum (ER)-replicating virus, as a model and systematically investigated the composition of BBSV VRCs in Nicotiana benthamiana by fusing the TurboID enzyme to viral replication protein p23. Among the 185 identified p23-proximal proteins, the reticulon family of proteins showed high reproducibility in the mass spectrometry data sets. We focused on RETICULON-LIKE PROTEIN B2 (RTNLB2) and demonstrated its proviral functions in BBSV replication. We showed that RTNLB2 binds to p23, induces ER membrane curvature, and constricts ER tubules to facilitate the assembly of BBSV VRCs. Our comprehensive proximal interactome analysis of BBSV VRCs provides a resource for understanding plant viral replication and offers additional insights into the formation of membrane scaffolds for viral RNA synthesis. TurboID-based proximity labeling reveals the viral replication complex structure of a plant virus, unveiling a proviral function of RETICULON-LIKE PROTEIN B2 in viral replication complex formation.

There are at least 400 types of human papillomaviruses (HPV) have been identified. HPVs infection is the most sexually transmitted disease in the US. Most of the HPVs are commensal or low-risk HPVs that cause either asymptomatic infection or benign skin and genital warts. However, about 17 types high–risk HPVs are the etiological agents responsible for 99% of cervical cancers and 5% of all human cancers worldwide. Although the vaccine is available, the protection is not full spectrum, and access to the vaccine is limited in some regions. In addition, condom use is not always effective to prevent HPV transmission. Thus, HPV is not a defeated pathogen, and it poses a significant burden on global health. HPVs are non-enveloped DNA viruses comprised of major capsid protein L1 and minor capsid protein L2. HPVs infect differentiating skin and mucosal tissues. HPV infection requires the viral particle to access the basal layer keratinocytes which sit atop the extracellular matrix-rich basement membrane. From there, virion particles will bind to the cell surface followed by endocytosis to enter the host cell. L2 is associated with viral genome (vDNA) and will act as an inducible transmembrane protein to protrude from endosome membrane and mediate the retrograde trafficking and nuclear importation of the viral genome to complete the initial infection. L2 itself and many cellular factors play an important role in these processes, but the detailed mechanisms between viral-host interaction and the L2 biology during infection are not well characterized. To address this knowledge gap, our overarching hypothesis is that after EGFR-dependent viral entry, the conserved L2 N-terminus region plays an important role in penetrating endosomal membrane while cellular SNX-BARs regulate viral retrograde trafficking. SNX-BAR proteins are likely involved with efficient entry as well. We further postulate that furin cleaved L2s may adopt a unique conformation, favorable for L2 membrane penetra

The machinery that decorates autophagic membranes with lipid-conjugated LC3/GABARAP is not yet fully understood. We recently reported the purification of the full-length ATG12-ATG5-ATG16L1 complex, and in reconstitution experiments with purified ATG7, ATG3, and LC3/GABARAP in vitro, together with rescue experiments in knockout cells, important aspects of the complete lipidation reaction were revealed. Hitherto unobserved membrane-binding regions in ATG16L1 were found, contributing to properties that explain the crucial role of this protein in membrane targeting and LC3/GABARAP lipidation in macroautophagy/autophagy and other related processes.

The machinery that decorates autophagic membranes with lipid-conjugated LC3/GABARAP is not yet fully understood. We recently reported the purification of the full-length ATG12-ATG5-ATG16L1 complex, and in reconstitution experiments with purified ATG7, ATG3, and LC3/GABARAP in vitro, together with rescue experiments in knockout cells, important aspects of the complete lipidation reaction were revealed. Hitherto unobserved membrane-binding regions in ATG16L1 were found, contributing to properties that explain the crucial role of this protein in membrane targeting and LC3/GABARAP lipidation in macroautophagy/autophagy and other related processes.

Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses th, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses th, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo-and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.

Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses th, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo-and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.

The importance of curvature as a structural feature of biological membranes has been recognized for many years and has fascinated scientists from a wide range of different backgrounds. On the one hand, changes in membrane morphology are involved in a plethora of phenomena involving the plasma membrane of eukaryotic cells, including endo-and exocytosis, phagocytosis and filopodia formation. On the other hand, a multitude of intracellular processes at the level of organelles rely on generation, modulation, and maintenance of membrane curvature to maintain the organelle shape and functionality. The contribution of biophysicists and biologists is essential for shedding light on the mechanistic understanding and quantification of these processes. Given the vast complexity of phenomena and mechanisms involved in the coupling between membrane shape and function, it is not always clear in what direction to advance to eventually arrive at an exhaustive understanding of this important research area. The 2018 Biomembrane Curvature and Remodeling Roadmap of Journal of Physics D: Applied Physics addresses this need for clarity and is intended to provide guidance both for students who have just entered the field as well as established scientists who would like to improve their orientation within this fascinating area.

Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses th, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses th, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

Local curvature is a key driving force for spatial organization of cellular membranes, via a phenomenon known as membrane curvature sensing, where the binding energy of membrane associated macromolecules depends on the local membrane shape. However, the microscopic mechanisms of curvature sensing are not well understood. Molecular dynamics simulations offer a powerful complement to biochemical experiments, yet their contribution to the study of curvature sensing has been limited, due in part to the lack of efficient methods, not least because of methodological difficulties in dealing with curved membranes. We develop a method based on simulated buckling, which has been previously employed to study mechanical properties of membranes. Here, we describe, validate and evaluate this method. We then apply to study curvature sensing properties of three model systems, using coarse-grained simulations. On the first system, we study lipid sorting in a three-component lipid mixture with emphasis on cardiolipin. We find that if curvature is high, curvature sensing is strong enough to drive cardiolipin molecules to negative curvature regions, outcompeting other lipids, without the need of external interactions or cooperative effects. We then simulated three systems consisting of a short amphipathic peptide attached to the surface of a buckled membrane. All three peptides localize to positive curvature, in agreement with the so-called cylindrical hydrophobic insertion mechanism. Their orientational preferences, however, defy the prediction of alignment perpendicular to the direction of maximum curvature. They also fail to show expected symmetries, indicating there is more to the picture than purely shape-based effects. The curvature sensing probe of the next system is a transmembrane trimeric protein, which shows preference to intermediate curvature, in agreement with theoretical predictions. But the lack of an expected 2-fold rotation symmetry indicates that the trimer senses th, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

The shape and composition of a membrane directly regulate the localization, activity, and signaling properties of membrane associated proteins. Proteins that both sense and generate membrane curvature, e.g., through amphiphilic insertion motifs, potentially engage in recursive binding dynamics, where the recruitment of the protein itself changes the properties of the membrane substrate. Simple geometric models of membrane curvature interactions already provide prediction tools for experimental observations, however these models are treating curvature sensing and generation as separated phenomena. Here, we outline a model that applies both geometric and basic thermodynamic considerations. This model allows us to predict the consequences of recursive properties in such interaction schemes and thereby integrate the membrane as a dynamic substrate. We use this combined model to hypothesize the origin and properties of tubular carrier systems observed in cells. Furthermore, we pinpoint the coupling to a membrane reservoir as a factor that influences the membrane curvature sensing and generation properties of local curvatures in the cell in line with classic determinants such as lipid composition and membrane geometry

The shape and composition of a membrane directly regulate the localization, activity, and signaling properties of membrane associated proteins. Proteins that both sense and generate membrane curvature, e.g., through amphiphilic insertion motifs, potentially engage in recursive binding dynamics, where the recruitment of the protein itself changes the properties of the membrane substrate. Simple geometric models of membrane curvature interactions already provide prediction tools for experimental observations, however these models are treating curvature sensing and generation as separated phenomena. Here, we outline a model that applies both geometric and basic thermodynamic considerations. This model allows us to predict the consequences of recursive properties in such interaction schemes and thereby integrate the membrane as a dynamic substrate. We use this combined model to hypothesize the origin and properties of tubular carrier systems observed in cells. Furthermore, we pinpoint the coupling to a membrane reservoir as a factor that influences the membrane curvature sensing and generation properties of local curvatures in the cell in line with classic determinants such as lipid composition and membrane geometry

The influenza A M2 protein is a 97-residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full-length protein. Here, the structure of the C-terminal juxtamembrane region (sites 50-60) is investigated in the full-length M2 protein using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50-60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C-terminal juxtamembrane region in full-length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full-length and truncated form of the protein.

The integral membrane proteins of the DP1 (deleted in polyposis) and reticulon families are responsible for maintaining the high membrane curvature required for both smooth endoplasmic reticulum (ER) tubules and the edges of ER sheets, and mutations in these proteins lead to motor neuron diseases, such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain reticulon homology domains (RHDs) that have unusually long hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilize membrane curvature. We have characterized the secondary structure and dynamics of the DP1 family protein produced from the YOP1 gene (Yop1p) and identified a C-terminal conserved amphipathic helix (APH) that, on its own, interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming APHs. Together, these results indicate that APHs play a previously unrecognized role in RHD membrane curvature stabilization.

Human ether-a-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in S4-S5 linker. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified a

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified a

In the first part of the thesis, protein-ligand interactions were investigated using the chaperone LcrH, from Yersinia as target protein. The structure of a peptide encompassing the amphipathic domain (residue 278-300) of the protein YopD from Yersinia was determined by NMR in 40% TFE. The structure of YopD278-300 is a well defined α-helix with a β-turn at the C-terminus of the helix capping the structure. This turn is crucial for the structure as peptides lacking the residues involved in the turn are unstructured. NMR relaxation indicates that the peptide is not monomeric. This is supported by intermolecular NOEs found from residue Phe280 to Ile288 and Val292 indicative of a multimeric structure with the helical structures oriented in an antiparallel manner with hydrophobic residues forming the oligomer. The interaction with the chaperone LcrH was confirmed by 1H relaxation experiments and induced chemical shift changes in the peptide Protein-ligand interactions were investigated further in the second paper using a different approach. A wide range of substances were used in screening for affinity against the chaperones PapD and FimC from uropathogenic Escherichia coli using 1H relaxation NMR experiments, surface plasmon resonance and 19F NMR. Fluorine NMR proved to be advantageous as compared to proton NMR as it is straight forward to identify binding ligands due to the well resolved 19F NMR spectra. Several compounds were found to interact with PapD and FimC through induced line-broadening and chemical shift changes for the ligands. Data corroborate well with surface plasmon resonance and proton NMR experiments. However, our results indicate the substances used in this study to have poor specificity for PapD and FimC as the induced chemical shift is minor and hardly no competitive binding is observed. Paper III and IV is an investigation of the structural features of the allergenic 2S albumin Ber e 1 from Brazil nut. Ber e 1 is a 2S albumin previously identified a