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Tumor heterogeneity remains a significant obstacle in cancer therapy due to diverse cells with varying treatment responses. Cancer stem-like cells (CSCs) contribute significantly to intratumor heterogeneity, characterized by high tumorigenicity and chemoresistance. CSCs reside in the depth of the tumor, possessing low reactive oxygen species (ROS) levels and robust antioxidant defense systems to maintain self-renewal and stemness. A nanotherapeutic strategy is developed using tumor-penetrating peptide iRGD-modified high-density lipoprotein (HDL)-mimetic nanodiscs (IPCND) that ingeniously loaded with pyropheophorbide-a (Ppa), bis (2-hydroxyethyl) disulfide (S-S), and camptothecin (CPT) by synthesizing two amphiphilic drug-conjugated sphingomyelin derivatives. Photoactivatable Ppa can generate massive ROS which as intracellular signaling molecules effectively shut down self-renewal and trigger differentiation of the CSCs, while S-S is utilized to deplete GSH and sustainably imbalance redox homeostasis by reducing ROS clearance. Simultaneously, the depletion of GSH is accompanied by the release of CPT, which leads to subsequent cell death. This dual strategy successfully disturbed the redox equilibrium of CSCs, prompting their differentiation and boosting the ability of CPT to kill CSCs upon laser irradiation. Additionally, it demonstrated a synergistic anti-cancer effect by concurrently eliminating therapeutically resistant CSCs and bulk tumor cells, effectively suppressing tumor growth in CSC-enriched heterogeneous colon tumor mouse models. © 2024 Wiley-VCH GmbH.

Proteins are the macromolecular machines responsible for many of life’s fundamental processes. As extended polymers of amino acids, proteins fold into unique three-dimensional shapes in order to carry out their biological roles. Understanding the connection between protein structure and function has been an active area of research for much of the last century, and throughout that time the primary method of structure determination has been X-ray crystallography. Recent advances in the field of cryo-electron microscopy (cryoEM), particularly with respect to electron detectors and algorithms for image processing, have allowed the technique to reach resolutions that rival X-ray diffraction studies. Without the requirement of a crystallized sample, cryoEM opens the possibility of visualizing previously intractable protein targets as well as flexibility and motion within those proteins. My thesis focuses on the application of single particle cryoEM to study membrane proteins in lipidic environments and the conformational changes they undergo. In the first chapter, I present a series of structures of the Bos taurus multidrug resistance protein 4 (MRP4). MRP4 is an ATP binding cassette transporter belonging to the ABCC family and is responsible for the rapid efflux of multiple endogenous and exogenous substrates. Included among these substrates are prostaglandins, a group of biologically active lipid molecules tied to physiological processes as diverse as inflammation and vasoactivity, and whose dysregulation is implicated in pathologies such as cancer and thrombosis. While MRP4 plays a crucial role in localized cell-to-cell signaling as the sole prostaglandin exporter identified in eukaryotes, the molecular details of it’s transport activity are poorly understood. Using single particle cryoEM, I determined five high resolution structures of MRP4 along various steps of the substrate transport cycle. These structures reveal for the first time the basis of MRP4’s affinity for

Tumor infarction therapy is a promising antitumor strategy with the advantages of taking a short therapy duration, less risk of resistance, and effectiveness against a wide range of tumor types. However, its clinical application is largely hindered by tumor recurrence in the surviving rim and the potential risk of thromboembolic events due to nonspecific vasculature targeting. Herein, a neovasculature-targeting synthetic high-density lipoprotein (sHDL) nanodisc loaded with pyropheophorbide-a and camptothecin (CPN) was fabricated for photoactivatable tumor infarction and synergistic chemotherapy. By manipulating the anisotropy in ligand modification of sHDL nanodiscs, CPN modified with neovaculature-targeting peptide on the planes (PCPN) shows up to 7-fold higher cellular uptake compared with that around the edge (ECPN). PCPN can efficiently bind to endothelial cells of tumor vessels, and upon laser irradiation, massive local thrombus can be induced by the photodynamic reaction to deprive nutrition supply. Meanwhile, CPT could be released in response to the tumor reductive environment, thus killing residual tumor cells in the surviving rim to inhibit recurrence. These findings not only offer a powerful approach of synergistic cancer therapy but also suggest the potential of plane-modified sHDL nanodiscs as a versatile drug delivery nanocarrier.

Viroporins are a class of viral membrane proteins that play diverse roles in the viral infection cycle. Viroporins are known to play roles in ion transport, inducing membrane curvature, as well as participating in a variety of protein-protein interactions. However, there is little known about the oligomeric state of viroporins. The lack of quantitative data on the oligomeric state of viroporins is due to key analytical challenges associated viroporins, including that they are 1) hydrophobic, and require a membrane mimetic for analysis, 2) small, making viroporins unamenable to some structural biology techniques such as cryo-EM, and 3) contain intrinsically disordered regions, which also poses challenges for structural characterization. To overcome these analytical challenges, this dissertation outlines the application of the unique combination of native mass spectrometry (MS) and nanodiscs for the oligomeric state determination of viroporins. The unique combination of native MS and nanodiscs can be used to determine the oligomeric state and lipid specificities of membrane proteins (such as viroporins) while embedded within a lipid bilayer using mass defect analysis. This dissertation reviews how to perform mass defect analysis, provides examples of how mass defect analysis has been applied in the past, as well as providing tips for overcoming some of the technical limitations associated with mass defect analysis. This dissertation also outlines a structure-activity relationship study performed to identify novel charge reducing agents for native MS. Charge reducing agents can be useful for the preservation of fragile complexes, such as viroporins, during native MS. Here, we found improved charge reducing agents that were effective on a wide range of analytes. We also uncovered the chemical principles governing charge reduction, which may provide a basis for the development of even better novel charge reagents in the future. The first viroporin characterized with nati

Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, Yersinia pestis type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).

Cardiovascular disease, the leading cause of mortality worldwide, is primarily caused by atherosclerosis, which is characterized by lipid and inflammatory cell accumulation in blood vessels and carotid intima thickening. Although disease management has improved significantly, new therapeutic strategies focused on accelerating atherosclerosis regression must be developed. Atherosclerosis models mimicking in vivo-like conditions provide essential information for research and new advances toward clinical application. New nanotechnology-based therapeutic opportunities have emerged with apoA-I nanoparticles (recombinant/reconstituted high-density lipoproteins, rHDL) as ideal carriers to deliver molecules and the discovery that microRNAs participate in atherosclerosis establishment and progression. Here, a therapeutic strategy to improve cholesterol efflux is developed based on a two-step administration of rHDL consisting of a first dose of antagomiR-33a-loaded rHDLs to induce adenosine triphosphate-binding cassette transporters A1 overexpression, followed by a second dose of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine rHDLs, which efficiently remove cholesterol from foam cells. A triple-cell 2D-atheroma plaque model reflecting the cellular complexity of atherosclerosis is used to improve efficiency of the nanoparticles in promoting cholesterol efflux. The results show that sequential administration of rHDL potentiates cholesterol efflux indicating that this approach may be used in vivo to more efficiently target atherosclerotic lesions and improve prognosis of the disease.

Supported lipid bilayers (SLBs) are commonly used as model systems mimicking biological membranes. Recently, we reported a new method to produce SLBs with incorporated membrane proteins, which is based on the application of peptide discs [Luchini et al., Analytical Chemistry, 2020, 92, 1081-1088]. Peptide discs are small discoidal particles composed of a lipid core and an outer belt of self-assembled 18A peptides. SLBs including membrane proteins can be formed by depositing the peptide discs on a solid support and subsequently removing the peptide by buffer rinsing. Here, we introduce a new variant of the 18A peptide, named dark peptide (d18A). d18A exhibits UV absorption at 214 nm, whereas the absorption at 280 nm is negligible. This improves sample preparation as it enables a direct quantification of the membrane protein concentration in the peptide discs by measuring UV absorption at 280 nm. We describe the application of the peptide discs prepared with d18A (dark peptide discs) to produce SLBs with a membrane protein, synaptobrevin 2 (VAMP2). The collected data showed the successful formation of SLBs with high surface coverage and incorporation of VAMP2 in a single orientation with the extramembrane domain exposed towards the bulk solvent. Compared to 18A, we found that d18A was more efficiently removed from the SLB. Our data confirmed the structural organisation of VAMP2 as including both alpha-helical and beta-sheet secondary structure. We further verified the orientation of VAMP2 in the SLBs by characterising the binding of VAMP2 with alpha-synuclein. These results point at the produced SLBs as relevant membrane models for biophysical studies as well as nanostructured biomaterials.

Natural cell membranes are diverse and complex amphipathic barriers with thousands of different lipids that play multiple physiological functions. How membrane protein structure and function are affected by different lipids in the cell membrane is understudied. Furthermore, the lipid bilayer composition can change with age, disease, and diet;how these changes affect membrane protein structure and their link to disease and pathology are unknown. Measuring how different lipid interactions affect membrane protein and peptide structure is challenging because they require a hydrophobic lipid environment to be stable. To address this challenge, this dissertation details methods to assemble lipid bilayer mimetics called nanodiscs with multiple lipids that are resolvable by native mass spectrometry (MS). This allows us to retain noncovalent interactions and measure the oligomeric state of incorporated peptides or proteins in intact mixed lipid nanodiscs. Furthermore, these methods are applied to the characterization of gene therapy delivery systems termed adeno-associated viral (AAV) capsids.Previously, native MS of nanodiscs was limited to one or two lipid systems because of the added polydispersity in the mass spectra caused by the addition of a new lipid. To resolve nanodiscs with multiple incorporated lipids, we chose lipids that were integer or fraction multiples of each other, so that their m/z peaks overlap constructively. Using this mass resonance approach, we resolved two lipid nanodisc systems with sterols, monosialotetrahexosyl-ganglioside (GM1), and cardiolipin. Then, we assembled and resolved model mammalian, bacterial, and mitochondrial nanodiscs with up to 4 different phospholipids, which were useful for characterizing polydisperse lipid interactions with LL-37, which is a potential therapeutic for antibiotic resistant bacteria. Moving to more complex lipid systems, we investigated nanodiscs made from commercially available natural lipid extract that provide

Antimicrobial peptides (AMPs) are an important part of the innate immune system and demonstrate promising applications in the fight against antibiotic resistant infections due to their unique mechanism of targeting bacterial membranes. Nevertheless, it is challenging to study the interactions of these peptides within lipid bilayers, making it difficult to understand their mechanisms of toxicity and selectivity. Lipoprotein nanodiscs are ideally suited for the study of AMPs with native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane peptides into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. This dissertation first describes a method to simplify interpretation of nanodisc spectra using an engineered series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, thus eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Next, this dissertation describes the use of fast photochemical oxidation of peptides (FPOP), an irreversible footprinting technique that labels solvent accessible residues, in combination with LC-MS-MS and native charge detection-mass spectrometry to study AMP-lipid interactions and surface exposed residues within different lipid bilayer nanodiscs. The findings herein provide complementary information on the potential modes of action and lipid selectivity of various AMPs.

Membrane proteins constitute a significant fraction of cellular proteins due to their vast diversity in function. However, studies of their native structures, folding mechanisms, and dynamics remain a challenge, in part because of their inherent insolubility. One goal of this project is to enhance the tools to study membrane proteins. Small unilamellar vesicles (SUVs) have been used as a bilayer mimetic to investigate membrane protein structures, dynamics, and folding mechanisms. UV resonance Raman spectroscopy (UVRR) has emerged as a powerful technique for probing structures and dynamics of biomolecules. UVRR is particularly valuable for membrane proteins because of the selective enhancement of signal from key aromatic residues and the ability to probe interactions between membrane protein and the lipid. We present practical considerations and guidelines for UVRR data acquisition, including a detailed description of a typical laser setup as well as the process to analyze the tryptophan vibrational modes of a model β-barrel membrane protein, OmpA, unfolded and folded in SUVs. An additional need in studies of membrane protein folding is alternate membrane mimics. Nanodiscs (NDs) are an excellent bilayer alternative to SUVs because of their experimental benefits including homogeneity, optical clarity, low light scattering, and enhanced stability. We combine SDS-PAGE mobility studies with fluorescence, circular dichroism, and UVRR spectroscopy to confirm and characterize the folding of OmpA into NDs. Our studies show similar secondary and tertiary structures in both SUVs and NDs as well as efficient folding yields greater than 88% in both bilayers. The folding of OmpA into NDs was slower compared to in SUVs, and this difference can be attributed to the different bilayer characteristics. Insights into the folding mechanism of OmpA were gained via bimolecular fluorescence quenching with acrylamide quencher. Stern-Volmer analysis utilizing the sphere-of-action model probe

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

New software, called Marbles, is introduced that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the contribution of the nanodisc to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as a set of beads, similarly to as in well known ab initio methods. The reliability and flexibility of this approach is proved by benchmarking the code, implemented in C++ with a Python interface, on a toy model and two proteins with very different geometry and size.

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

Membrane proteins (MPs) constitute a large fraction of the proteome, but exhibit physicochemical characteristics that impose challenges for successful sample production crucial for subsequent biophysical studies. In particular, MPs have to be extracted from the membranes in a stable form. Reconstitution into detergent micelles represents the most common procedure in recovering MPs for subsequent analysis. n-dodecyl-beta-D-maltoside (DDM) remains one of the most popular conventional detergents used in production of MPs. Here we characterize the novel DDM analogue 4-trans-(4-trans-propylcyclohexyl)-cyclohexyl alpha-maltoside (t-PCC alpha M), possessing a substantially lower critical micelle concentration (CMC) than the parental compound that represents an attractive feature when handling MPs. Using three different types of MPs of human and prokaryotic origin, i. e., a channel, a primary and a secondary active transporter, expressed in yeast and bacterial host systems, respectively, we investigate the performance of t-PCC alpha M in solubilization and affinity purification together with its capacity to preserve native fold and activity. Strikingly, t-PCC alpha M displays favorable behavior in extracting and stabilizing the three selected targets. Importantly, t-PCC alpha M promoted extraction of properly folded protein, enhanced thermostability and provided negatively-stained electron microscopy samples of promising quality. All-in-all, t-PCC alpha M emerges as competitive surfactant applicable to a broad portfolio of challenging MPs for downstream structure-function analysis.

New software, called Marbles, is introduced that employs SAXS intensities to predict the shape of membrane proteins embedded into membrane nanodiscs. To gain computational speed and efficient convergence, the strategy is based on a hybrid approach that allows one to account for the contribution of the nanodisc to the SAXS intensity through a semi-analytical model, while the embedded membrane protein is treated as a set of beads, similarly to as in well known ab initio methods. The reliability and flexibility of this approach is proved by benchmarking the code, implemented in C++ with a Python interface, on a toy model and two proteins with very different geometry and size.

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

Membrane proteins (MPs) constitute a large fraction of the proteome, but exhibit physicochemical characteristics that impose challenges for successful sample production crucial for subsequent biophysical studies. In particular, MPs have to be extracted from the membranes in a stable form. Reconstitution into detergent micelles represents the most common procedure in recovering MPs for subsequent analysis. n-dodecyl-beta-D-maltoside (DDM) remains one of the most popular conventional detergents used in production of MPs. Here we characterize the novel DDM analogue 4-trans-(4-trans-propylcyclohexyl)-cyclohexyl alpha-maltoside (t-PCC alpha M), possessing a substantially lower critical micelle concentration (CMC) than the parental compound that represents an attractive feature when handling MPs. Using three different types of MPs of human and prokaryotic origin, i. e., a channel, a primary and a secondary active transporter, expressed in yeast and bacterial host systems, respectively, we investigate the performance of t-PCC alpha M in solubilization and affinity purification together with its capacity to preserve native fold and activity. Strikingly, t-PCC alpha M displays favorable behavior in extracting and stabilizing the three selected targets. Importantly, t-PCC alpha M promoted extraction of properly folded protein, enhanced thermostability and provided negatively-stained electron microscopy samples of promising quality. All-in-all, t-PCC alpha M emerges as competitive surfactant applicable to a broad portfolio of challenging MPs for downstream structure-function analysis.

Membrane proteins play vital roles in many biological processes and represent the majority of drug targets. The structural and functional integrity of membrane proteins can be heavily influenced by their surrounding lipid environment. However, membrane protein-lipid interactions are challenging to study in lipid bilayers due to their transient and polydisperse nature. Nanodiscs provide a versatile and robust membrane system for solubilizing membrane proteins in a native-like lipid bilayer. This dissertation describes the application of nanodiscs and native mass spectrometry to investigate the selectivity of membrane protein-lipid interactions. Charge manipulation additives and electrospray ionization conditions described in this dissertation allow membrane protein nanodiscs to be stabilized or destabilized during native mass spectrometry. Supercharging reagents in negative mode stabilized nanodiscs containing the ammonium transporter (AmtB) or aquaporin (ApqZ) and facilitated the measurement of membrane protein oligomeric state within an intact lipid bilayer. Conversely, supercharging reagents in positive mode ejected AmtB and AqpZ with tightly bound lipids, allowing the measurement of more specific interactions. To study how AmtB remodels its surrounding lipid environment, native mass spectrometry was performed for AmtB-lipid complexes following gas-phase ejection or solution-phase detergent extraction. Studies showed that AmtB is selective overall for POPG lipids but is enriched in POPC for tightly bound lipids. The mass spectrometry approaches detailed in this dissertation provide novel directions in using nanodiscs for characterizing membrane protein-lipid interactions in heterogeneous systems and are promising for applying nanodiscs in therapeutic development.

Analysis of proteins and complexes under native mass spectrometric (MS) and solution conditions was typically performed using time-of-flight (ToF) analyzers, due to their routine high m/z transmission and detection capabilities. However, over recent years, the ability of Orbitrap-based mass spectrometers to transmit and detect a range of high molecular weight species is well documented. Herein, we describe how a 15 Tesla Fourier transform ion cyclotron resonance mass spectrometer (15 T FT-ICR MS) is more than capable of analyzing a wide range of ions in the high m/z scale (>5000), in both positive and negative instrument polarities, ranging from the inorganic cesium iodide salt clusters; a humanized IgG1k monoclonal antibody (mAb; 148.2 kDa); an IgG1-mertansine drug conjugate (148.5 kDa, drug-to-antibody ratio; DAR 2.26); an IgG1-siRNA conjugate (159.1 kDa; ribonucleic acid to antibody ratio; RAR 1); the membrane protein aquaporin-Z (97.2 kDa) liberated from a C8E4 detergent micelle; the empty MSP1D1-nanodisc (142.5 kDa) and the tetradecameric chaperone protein complex GroEL (806.2 kDa; GroEL dimer at 1.6 MDa). We also investigate different regions of the FT-ICR MS that impact ion transmission and desolvation. Finally, we demonstrate how the transmission of these species and resultant spectra are highly consistent with those previously generated on both quadrupole-ToF (Q-ToF) and Orbitrap instrumentation. This report serves as an impactful example of how FT-ICR mass analyzers are competitive to Q-ToFs and Orbitraps for high mass detection at high m/z.

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study. Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR. Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this pro, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study. Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR. Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this pro, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study. Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR. Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this pro, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Understanding the structure-function relationship of membrane receptors is essential to comprehend the crosstalk between key signaling pathways. Aberrant trans-activation between receptors can lead to tumorigenesis. Two of these receptors known to be involved in cancer development are receptor tyrosine kinases (RTKs), RON (Recepteur d'Origine Nantais) and EGFR (Epidermal Growth Factor Receptor). There has been evidence of heterodimerization and crosstalk between these two receptors based on co-immunoprecipitation, however the structural requirements behind these interactions remain unknown. Structural studies could provide insights into these RTKs’ modes of dimerization and structure-function relationship. However, structural studies of full-length membrane proteins are often difficult due to poor solubility of the hydrophobic transmembrane domains. This affects protein structure and functionality. The use of nanodiscs for protein structural studies helps provide a native-like environment for membrane proteins, helping to avoid denaturing and aggregation, as well as providing a homogeneous size which makes them ideal for imaging techniques. In this work, we focus on optimizing a nanodisc assembly protocol to incorporate full-length RON and EGFR receptors into nanodiscs, as well as developing techniques to detect protein incorporation into these nanodiscs, which would ultimately facilitate structural studies for RON and EGFR heterodimerization. These studies could provide a mechanistic justification for novel targeted therapies in cancer.

Understanding the structure-function relationship of membrane receptors is essential to comprehend the crosstalk between key signaling pathways. Aberrant trans-activation between receptors can lead to tumorigenesis. Two of these receptors known to be involved in cancer development are receptor tyrosine kinases (RTKs), RON (Recepteur d'Origine Nantais) and EGFR (Epidermal Growth Factor Receptor). There has been evidence of heterodimerization and crosstalk between these two receptors based on co-immunoprecipitation, however the structural requirements behind these interactions remain unknown. Structural studies could provide insights into these RTKs’ modes of dimerization and structure-function relationship. However, structural studies of full-length membrane proteins are often difficult due to poor solubility of the hydrophobic transmembrane domains. This affects protein structure and functionality. The use of nanodiscs for protein structural studies helps provide a native-like environment for membrane proteins, helping to avoid denaturing and aggregation, as well as providing a homogeneous size which makes them ideal for imaging techniques. In this work, we focus on optimizing a nanodisc assembly protocol to incorporate full-length RON and EGFR receptors into nanodiscs, as well as developing techniques to detect protein incorporation into these nanodiscs, which would ultimately facilitate structural studies for RON and EGFR heterodimerization. These studies could provide a mechanistic justification for novel targeted therapies in cancer.

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study. Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR. Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this pro, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study. Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR. Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this pro, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study. Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (FeB) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for FeB cofactor insertion into cNOR. Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked FeB at the catalytic center but was otherwise structurally intact. Therefore we used this pro, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.

In this work we report on plasma oxidation of palladium (Pd) to form reliable palladium/palladium oxide (Pd/PdOx) core-shell sensor for ppb level H2S detection and its performance improvement through nanostructuring using hole-mask colloidal lithography (HCL). The plasma oxidation parameters and the sensor operating conditions are optimized to arrive at a sensor device with high sensitivity and repeatable response for H2S. The plasma oxidized palladium/palladium oxide sensor shows a response of 43.1% at 3 ppm H2S at the optimum operating temperature of 200 degrees C with response and recovery times of 24 s and 155 s, respectively. The limit of detection (LoD) of the plasma oxidised beam is 10 ppb. We further integrate HCL, a bottom-up and cost-effective process, to create nanodiscs of fixed diameter of 100 nm and varying heights (10, 15 and 20 nm) on 10 nm thin Pd beam which is subsequently plasma oxidized to improve the H2S sensing characteristics. The nanostructured Pd/PdOx sensor with nanodiscs of 100 nm diameter and 10 nm height shows an enhancement in sensing performance by 11.8% at same operating temperature and gas concentration. This nanostructured sensor also shows faster response and recovery times (15s and 100s, respectively) compared to the unstructured Pd/PdOx counterpart together with an experimental LoD of 10 ppb and the estimated limit going all the way down to 2 ppb. Material characterization of the fabricated Pd/PdOx sensors is done using UV-vis spectroscopy and x-ray photoemission spectroscopy., QC 20180515

Membrane proteins are notoriously difficult to investigate in isolation. The focus of this chapter is the key step following extraction and purification of membrane proteins; namely reconstitution. The process of reconstitution re-inserts proteins into a lipid bilayer that partly resembles their native environment. This native environment is vital to the stability of membrane proteins, ensuring that they undergo vital conformational transitions and maintain optimal interaction with their substrates. Reconstitution may take many forms and these have been classified into two broad categories. Symmetric systems enable unfettered access to both sides of a bilayer. Compartment containing systems contain a lumen and are ideally suited to measurement of transport processes. The investigator is encouraged to ascertain what aspects of protein function will be undertaken and to apply the most advantageous reconstitution system or systems. It is important to note that the process of reconstitution is not subject to defined protocols and requires empirical optimisation to specific targets.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.

The structure, dynamics, and function of membrane proteins are intimately linked to the properties of the membrane environment in which the proteins are embedded. For structural and biophysical characterization, membrane proteins generally need to be extracted from the membrane and reconstituted in a suitable membrane-mimicking environment. Ensuring functional and structural integrity in these environments is often a major concern. The styrene/maleic acid co-polymer has recently been shown to be able to extract lipid/membrane protein patches directly from native membranes to form nanosize discoidal proteolipid particles, also referred to as native nanodiscs. In this work, we show that high-resolution solid-state NMR spectra can be obtained from an integral membrane protein in native nanodiscs, as exemplified by the 2×34 kDa bacterial cation diffusion facilitator CzcD.

A promising tool in membrane research is the use of the styrene-maleic acid (SMA) copolymer to solubilize membranes in the form of nanodiscs. Since membranes are heterogeneous in composition, it is important to know whether SMA thereby has a preference for solubilization of either specific types of lipids or specific bilayer phases. Here, we investigated this by performing partial solubilization of model membranes and analyzing the lipid composition of the solubilized fraction. We found that SMA displays no significant lipid preference in homogeneous binary lipid mixtures in the fluid phase, even when using lipids that by themselves show very different solubilization kinetics. By contrast, in heterogeneous phase-separated bilayers, SMA was found to have a strong preference for solubilization of lipids in the fluid phase as compared to those in either a gel phase or a liquid-ordered phase. Together the results suggest that (1) SMA is a reliable tool to characterize native interactions between membrane constituents, (2) any solubilization preference of SMA is not due to properties of individual lipids but rather due to properties of the membrane or membrane domains in which these lipids reside and (3) exploiting SMA resistance rather than detergent resistance may be an attractive approach for the isolation of ordered domains from biological membranes.

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificansmembrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O2-binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O2, and found that the pKa of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed.

Autophosphorylating histidine kinase CheA is central to signaling in bacterial chemotaxis. The kinase donates its phosphoryl group to two response regulators, CheY that controls flagellar rotation and thus motility and CheB, crucial for sensory adaptation. As measured by coupled CheY phosphorylation, incorporation into signaling complexes activates the kinase ∼1000-fold and places it under control of chemoreceptors. By the same assay, receptors modulate kinase activity ∼100-fold as a function of receptor ligand occupancy and adaptational modification. These changes are the essence of chemotactic signaling. Yet, the enzymatic properties affected by incorporation into signaling complexes, by chemoreceptor ligand binding or by receptor adaptational modification are largely undefined. To investigate, we performed steady-state kinetic analysis of autophosphorylation using a liberated kinase phosphoryl-accepting domain, characterizing kinase alone, in isolated core signaling complexes and in small arrays of core complexes assembled in vitro with receptors contained in isolated native membranes. Autophosphorylation in signaling complexes was measured as a function of ligand occupancy and adaptational modification. Activation by incorporation into signaling complexes and modulation in complexes by ligand occupancy and adaptational modification occurred largely via changes in the apparent catalytic rate constant (kcat ). Changes in the autophosphorylation kcat accounted for most of the ∼1000-fold kinase activation in signaling complexes observed for coupled CheY phosphorylation, and the ∼100-fold inhibition by ligand occupancy or modulation by adaptational modification. Our results indicate no more than a minor role in kinase control for simple sequestration of the autophosphorylation substrate. Instead they indicate direct effects on the active site.

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitnficans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O-2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O-2-binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O-2, and found that the pK(a) of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed.

ECF (Energy-Coupling Factor)-Transporter sind prokaryotische Aufnahmesysteme für Mikronährstoffe, die eine spezielle Gruppe von Transportern mit ATP-Bindekassette (ABC) darstellen. Sie beinhalten zwei asymmetrische Membranproteine, von denen eins (S) für die spezifische Bindung und Translokation des Substrates und das andere (T) für die Kopplung mit den ATPasen (A1,A2) zuständig ist. Bei ECF-Transportern der Subklasse I bilden diese Komponenten eine Einheit, während bei Vertretern der Subklasse II ein AAT-Modul mit wechselnden S-Einheiten interagiert. In der vorliegenden Arbeit wurde der Transportmechanismus, der eine Drehung der kompletten S-Einheit in der Membran beinhaltet, anhand des Biotintransporters BioMNY erstmals experimentell validiert. Durch Rekonstitution in Lipid-Nanodiscs, chemische Quervernetzung, fluoreszenz- und ESR-spektroskopische Techniken sowie einen Bindungstest mit radioaktivem Biotin wurde gezeigt, dass (i) die ATP-Bindung an die ATPasen zu einer Aufrichtung der S-Einheit (BioY) führt, (ii) diese Bewegung die Substratbeladung ermöglicht und (iii) BioY dabei ununterbrochen mit der T-Einheit (BioN) interagiert. Dies stellt einen Gegensatz zu Systemen der Subklasse II dar, für die ein ATP-abhängiger Austausch von S-Einheiten im Transportzyklus gezeigt worden war. Darüber hinaus wurde ein Escherichia coli-Stamm konstruiert, der durch Blockierung seines hochaffinen Biotintransporters und des -synthesewegs auf Spuren von Biotin nicht wachsen kann. Dieser Stamm ermöglichte einen eindeutigen Nachweis der Transportaktivität einiger solitärer BioY-Proteine. Aufgrund der einheitlichen Topologie von S-Einheiten ist ein Kippen auch für solitäre BioY-Varianten wahrscheinlich. Auch die metallspezifischen S-Einheiten CbiM und NikM besitzen ohne AAT-Modul eine basale Co2+- bzw. Ni2+-Transportaktivität. Ein ESR-spektroskopischer Kobaltnachweises zeigte, dass die aus nur zwei Membranhelices bestehende CbiN-Einheit für die Metallbeladung von CbiM essentiell ist., ECF (Energy-Coupling Factor) transporters are a subgroup of ABC transporters that mediate uptake of micronutrients into prokaryotic cells. In contrast to canonical ABC importers, ECF transporters comprise two unrelated membrane proteins, one of which is responsible for specific and high affinity substrate binding (S) and the other one constitutes the coupling component (T) between S and the cytosolic ABC-ATPases (A1,A2). Subclass I transporters consist of four dedicated components whereas in subclass II transporters, a central AAT-module may interact with various S units. The biotin specific subclass I ECF transporter BioMNY was used to experimentally verify the hitherto hypothetic transport mechanism, which involves a rotation of the S unit within the membrane. With a series of experiments including reconstitution of BioMNY into lipid nanodiscs, site-specific cross-linking, a substrate binding assay with radioactive biotin and both fluorescence and EPR spectroscopic techniques, the ATP-dependent rotation of BioY (S) as a prerequisite for substrate binding and release was shown for the first time for an ECF transporter. Unlike subclass II transporters, for which an ATP-dependent release of the S unit was proposed, BioY interacts continuously with BioN (T) during the transport cycle. In a second focus of the work, an Escherichia coli reporter strain for biotin transporters was constructed. Due to inactivation of both biotin synthesis and the intrinsic high affinity biotin transporter, this strain was not capable of growing on trace amounts of biotin. With the use of this strain, transport activity of recombinantly produced solitary BioY proteins that naturally lack other ECF components was evidenced. Transport activity in the absence of AAT modules is also a feature of the Co2+ and Ni2+ specific S components CbiM and NikM. An EPR spectroscopic Co2+ detection assay helped underscoring the essential role of the small membrane protein CbiN for Co2+ loading of CbiM.

Autophosphorylating histidine kinase CheA is central to signaling in bacterial chemotaxis. The kinase donates its phosphoryl group to two response regulators, CheY that controls flagellar rotation and thus motility and CheB, crucial for sensory adaptation. As measured by coupled CheY phosphorylation, incorporation into signaling complexes activates the kinase ∼1000-fold and places it under control of chemoreceptors. By the same assay, receptors modulate kinase activity ∼100-fold as a function of receptor ligand occupancy and adaptational modification. These changes are the essence of chemotactic signaling. Yet, the enzymatic properties affected by incorporation into signaling complexes, by chemoreceptor ligand binding or by receptor adaptational modification are largely undefined. To investigate, we performed steady-state kinetic analysis of autophosphorylation using a liberated kinase phosphoryl-accepting domain, characterizing kinase alone, in isolated core signaling complexes and in small arrays of core complexes assembled in vitro with receptors contained in isolated native membranes. Autophosphorylation in signaling complexes was measured as a function of ligand occupancy and adaptational modification. Activation by incorporation into signaling complexes and modulation in complexes by ligand occupancy and adaptational modification occurred largely via changes in the apparent catalytic rate constant (kcat ). Changes in the autophosphorylation kcat accounted for most of the ∼1000-fold kinase activation in signaling complexes observed for coupled CheY phosphorylation, and the ∼100-fold inhibition by ligand occupancy or modulation by adaptational modification. Our results indicate no more than a minor role in kinase control for simple sequestration of the autophosphorylation substrate. Instead they indicate direct effects on the active site.

Conformational equilibria of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here conformational substates of the GPCR rhodopsin are investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helices 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance spectroscopy. The photoactivated receptor in DDM is dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is observed, even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decays to that characteristic of the inactive state with a lifetime of ∼16 min at 20 °C. Importantly, transducin binding to the activated receptor selects a subset of the ensemble in which multiple substates are apparently retained. The results indicate that in a native-like lipid environment rhodopsin activation is not analogous to a simple binary switch between two defined conformations, but the activated receptor is in equilibrium between multiple conformers that in principle could recognize different binding partners.

ECF (Energy-Coupling Factor)-Transporter sind prokaryotische Aufnahmesysteme für Mikronährstoffe, die eine spezielle Gruppe von Transportern mit ATP-Bindekassette (ABC) darstellen. Sie beinhalten zwei asymmetrische Membranproteine, von denen eins (S) für die spezifische Bindung und Translokation des Substrates und das andere (T) für die Kopplung mit den ATPasen (A1,A2) zuständig ist. Bei ECF-Transportern der Subklasse I bilden diese Komponenten eine Einheit, während bei Vertretern der Subklasse II ein AAT-Modul mit wechselnden S-Einheiten interagiert. In der vorliegenden Arbeit wurde der Transportmechanismus, der eine Drehung der kompletten S-Einheit in der Membran beinhaltet, anhand des Biotintransporters BioMNY erstmals experimentell validiert. Durch Rekonstitution in Lipid-Nanodiscs, chemische Quervernetzung, fluoreszenz- und ESR-spektroskopische Techniken sowie einen Bindungstest mit radioaktivem Biotin wurde gezeigt, dass (i) die ATP-Bindung an die ATPasen zu einer Aufrichtung der S-Einheit (BioY) führt, (ii) diese Bewegung die Substratbeladung ermöglicht und (iii) BioY dabei ununterbrochen mit der T-Einheit (BioN) interagiert. Dies stellt einen Gegensatz zu Systemen der Subklasse II dar, für die ein ATP-abhängiger Austausch von S-Einheiten im Transportzyklus gezeigt worden war. Darüber hinaus wurde ein Escherichia coli-Stamm konstruiert, der durch Blockierung seines hochaffinen Biotintransporters und des -synthesewegs auf Spuren von Biotin nicht wachsen kann. Dieser Stamm ermöglichte einen eindeutigen Nachweis der Transportaktivität einiger solitärer BioY-Proteine. Aufgrund der einheitlichen Topologie von S-Einheiten ist ein Kippen auch für solitäre BioY-Varianten wahrscheinlich. Auch die metallspezifischen S-Einheiten CbiM und NikM besitzen ohne AAT-Modul eine basale Co2+- bzw. Ni2+-Transportaktivität. Ein ESR-spektroskopischer Kobaltnachweises zeigte, dass die aus nur zwei Membranhelices bestehende CbiN-Einheit für die Metallbeladung von CbiM essentiell ist., ECF (Energy-Coupling Factor) transporters are a subgroup of ABC transporters that mediate uptake of micronutrients into prokaryotic cells. In contrast to canonical ABC importers, ECF transporters comprise two unrelated membrane proteins, one of which is responsible for specific and high affinity substrate binding (S) and the other one constitutes the coupling component (T) between S and the cytosolic ABC-ATPases (A1,A2). Subclass I transporters consist of four dedicated components whereas in subclass II transporters, a central AAT-module may interact with various S units. The biotin specific subclass I ECF transporter BioMNY was used to experimentally verify the hitherto hypothetic transport mechanism, which involves a rotation of the S unit within the membrane. With a series of experiments including reconstitution of BioMNY into lipid nanodiscs, site-specific cross-linking, a substrate binding assay with radioactive biotin and both fluorescence and EPR spectroscopic techniques, the ATP-dependent rotation of BioY (S) as a prerequisite for substrate binding and release was shown for the first time for an ECF transporter. Unlike subclass II transporters, for which an ATP-dependent release of the S unit was proposed, BioY interacts continuously with BioN (T) during the transport cycle. In a second focus of the work, an Escherichia coli reporter strain for biotin transporters was constructed. Due to inactivation of both biotin synthesis and the intrinsic high affinity biotin transporter, this strain was not capable of growing on trace amounts of biotin. With the use of this strain, transport activity of recombinantly produced solitary BioY proteins that naturally lack other ECF components was evidenced. Transport activity in the absence of AAT modules is also a feature of the Co2+ and Ni2+ specific S components CbiM and NikM. An EPR spectroscopic Co2+ detection assay helped underscoring the essential role of the small membrane protein CbiN for Co2+ loading of CbiM.

Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge, and to date there is limited number of available functional assays. In this chapter, we describe in detail our recently developed methodology to reconstitute membrane proteins such as the integral membrane protein cytochrome P450 oxidoreductase on membrane systems such as Nanodiscs and study their functional dynamics by recordings at the fundamental resolution of individual catalytic turnovers using prefluorescent substrate analogues. We initially describe the methodology for reconstitution, surface immobilization, and data acquisition of individual enzyme catalytic turnovers. We then explain in detail the statistical analysis, with an emphasis on the model development, the potential pitfalls for correctly identifying the abundance, lifetime, and likelihood of sampling protein functional states. This methodology may enable studies of functional dynamics and their role in biology for a spectrum of membrane proteins.

Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches., Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches.

Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches., Soluble nanoscale lipid bilayers, termed nanodiscs, are widely used in science for studying the membrane-anchored and integral membrane protein complexes under defined experimental conditions. Although their formation occurs by a self-assembly process, nanodisc purification and the verification of proper reconstitution are still major challenges during the sample preparation. This review gives an overview of the methods used for purifying and analyzing nanodiscs and nanodisc-reconstituted membrane proteins, with an emphasis on the chromatographic and electrophoretic approaches.

Leukotrienes are specialized lipid molecules derived from arachidonic acid that have severe pathological roles in inflammatory diseases like asthma, allergy and in the formation of cardiovascular diseases. Since these diseases can be fatal if not treated, it is significant to study leukotriene products and how they form. The formation of leukotrienes from arachidonic acid is a multi step process. This event is aided by different enzymes. Two such enzymes are 5–lipoxygenase (5-LO) and 5-lipoxygenase activating protein (FLAP). FLAP is an integral membrane protein and belongs to the MAPEG super family (Membrane Associated proteins in Eicosanoid and Glutathione metabolism). In the leukotriene synthesis pathway, the only function of FLAP is to increase the oxygenation reaction by assisting the transfer of arachidonic acid from nuclear membrane to 5-LO. In fact, FLAP doesn’t seem to have any enzymatic/mechanistic activity on its own. Many biological questions in this pathway are unanswered. Is there any functional activity of FLAP? Is there any physical contact between FLAP and 5-LO in the leukotriene formation? To address these questions, we introduced a novel method of studying the pathway by constructing nanodiscs that are small soluble pieces of membrane of defined sizes. The aim of the project is to develop a protocol for generation of nanodiscs that contain FLAP (so called reconstitution of FLAP) and to verify the presence of FLAP in the nanodiscs by practical techniques such as blue native PAGE and Western Blot. Images from transmission electron microscopy were less conclusive but improvement can be done in future by use of antibodies to visualize FLAP nanodiscs. Ultimately, reconstitution of FLAP was successfully completed for further structural and functional studies. To conclude, this project paves way for the studies of complex formation of integral membrane proteins with soluble proteins and further applications on pharmaceutical front.

Kryštálovú štruktúru materiálov s fázovou premenou dokážeme meniť dodaním vonkajšej energie, čo má za následok zmenu ich elektrických alebo magnetických vlastností. Hoci sa tento efekt ovládania plazmonických rezonancií v nanofotonike využíva, dva materiály zvolené v tejto práci — oxid vanadičitý (VO2) a zliatina železa a ródia (FeRh) — neboli zatiaľ dostatočne preskúmané. Plazmonické rezonancie môžu byť charakterizované ako rezonancie electromagnetického poľa v kovovvých nanoštruktúrach. Týmito nanoštruktúrami sme dokonca schopní ovládať svetlo. Na začiatku tejto práce sa venujeme optimalizovaniu procesu elektrónovej litografie pre výrobu 50nm vysokých kovových nanodiskov s priemermi 40–200nm. V druhej časti skúmame optickú odozvu zlatých nanodiskov, aby sme lepšie pochopili povahu plazmonických rezonancií a interakcií medzi nimi. V poslednej časti sa venujeme optimalizácii výroby polykryštalického VO2 a meraniam optických odoziev VO2 a FeRh nanodiskov počas ich fázovej premeny. Pri meraní VO2 nanodiskov v dielektrickej fázi sme pozorovali Mieho rezonancie. Tieto nanodisky sa majú potenciál chovať ako laditeľné plazmonické štruktúry, ktorých Mieho rezonancie v dielektrickej fázy VO2 sa menia na plazmonické rezonancie v kovovej fázy. Počas merania FeRh nanodiskov sme pozorovali plazmonické rezonancie vo viditeľnom spektre. Tieto rezonancie môžu byť použité na zníženie energie potrebnej na prechod FeRh z anti-feromagnetickej do feromagnetickej fázy., The crystal structure of phase-change materials can be reconfigured by external stimuli, which often result in a change of materials electrical or magnetic properties. Although this effect has been already used to modulate plasmonic resonances in nanophotonics, it has not been fully examined for the two materials chosen in this work — vanadium dioxide (VO2) and iron-rhodium alloy (FeRh). Plasmonic resonances can be described as resonances of electromagnetic field in metallic nanostructures. With these nanostructures we are even able to modulate light. In this thesis, we firstly optimized electron beam lithography process for production of metal nanodiscs with 40–200nm diameters. Secondly, we measured an optical response of gold nanodiscs to better understand the nature of their plasmonic resonances and interactions between them. Lastly, we described the optimization of polycrystalline VO2 growth and measured optical responses of VO2 and FeRh nanodiscs during their respective phase transitions. Our observation of Mie’s resonances in the dielectric phase of the VO2 nanodiscs suggests, that they have a potential to act as tunable plasmonic resonators which switch from Mie’s resonances in the dielectric phase into plasmonic resonances in the metallic one. When measuring the FeRh nanodiscs, we observed plasmonic resonances in the visible part of the spectrum. These resonances can be used to facilitate FeRh transition from an anti-ferromagnetic to a ferromagnetic phase, as they could lower the required latent heat.