Molecular characterization of the conoid complex in Toxoplasma reveals its conservation in all apicomplexans, including Plasmodium species.

The apical complex is the instrument of invasion used by apicomplexan parasites, and the conoid is a conspicuous feature of this apparatus found throughout this phylum. The conoid, however, is believed to be heavily reduced or missing from Plasmodium species and other members of the class Aconoidasida. Relatively few conoid proteins have previously been identified, making it difficult to address how conserved this feature is throughout the phylum, and whether it is genuinely missing from some major groups. Moreover, parasites such as Plasmodium species cycle through 3 invasive forms, and there is the possibility of differential presence of the conoid between these stages. We have applied spatial proteomics and high-resolution microscopy to develop a more complete molecular inventory and understanding of the organisation of conoid-associated proteins in the model apicomplexan Toxoplasma gondii. These data revealed molecular conservation of all conoid substructures throughout Apicomplexa, including Plasmodium, and even in allied Myzozoa such as Chromera and dinoflagellates. We reporter-tagged and observed the expression and location of several conoid complex proteins in the malaria model P. berghei and revealed equivalent structures in all of its zoite forms, as well as evidence of molecular differentiation between blood-stage merozoites and the ookinetes and sporozoites of the mosquito vector. Collectively, we show that the conoid is a conserved apicomplexan element at the heart of the invasion mechanisms of these highly successful and often devastating parasites.

Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer-Ligand Interactions: From Mechanism to Binding Constants.

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found Kd1 = 69.7 µM and Kd2 = 5.3 µM for ABA (two binding sites); Kd1 = 22.04 µM for LABA; and Kd1 = 8.5 µM for CBA.

Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein-Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area.

Native electrospray ionization mass spectrometry (ESI-MS) is nowadays widely used for the direct and sensitive determination of protein complex stoichiometry and binding affinity constants ( Ka). A common yet poorly understood phenomenon in native ESI-MS is the difference between the charge-state distributions (CSDs) of the bound protein-ligand complex (PL) and unbound protein (P) signals. This phenomenon is typically attributed to experimental artifacts such as nonspecific binding or in-source dissociation and is considered highly undesirable, because the determined Ka values display strong variation with charge state. This situation raises serious concerns regarding the reliability of ESI-MS for the analysis of protein complexes. Here we demonstrate that, contrary to the common belief, the CSD difference between P and PL ions can occur without any loss of complex integrity, simply due to a change in the solvent-accessible surface area (ΔSASA) of the protein upon ligand binding in solution. The experimental CSD shifts for PL and P ions in ESI-MS are explained in relation to the magnitude of ΔSASA for diverse protein-ligand systems using a simple model based on the charged residue mechanism. Our analysis shows that the revealed ΔSASA factor should be considered rather general and be given attention for the correct spectral interpretation of protein complexes.

Dynamic Assembly and Disassembly of Functional ß-Endorphin Amyloid Fibrils.

Neuropeptides and peptide hormones are stored in the amyloid state in dense-core vesicles of secretory cells. Secreted peptides experience dramatic environmental changes in the secretory pathway, from the endoplasmic reticulum via secretory vesicles to release into the interstitial space or blood. The molecular mechanisms of amyloid formation during packing of peptides into secretory vesicles and amyloid dissociation upon release remain unknown. In the present work, we applied thioflavin T binding, tyrosine intrinsic fluorescence, fluorescence anisotropy measurements, and solid-state NMR spectroscopy to study the influence of physiologically relevant environmental factors on the assembly and disassembly of ß-endorphin amyloids in vitro. We found that ß-endorphin aggregation and dissociation occur in vitro on relatively short time scales, comparable to times required for protein synthesis and the rise of peptide concentration in the blood, respectively. Both assembly and disassembly of amyloids strongly depend on the presence of salts of polyprotic acids (such as phosphate and sulfate), while salts of monoprotic acids are not effective in promoting aggregation. A steep increase of the peptide aggregation rate constant upon increase of solution pH from 5.0 to 6.0 toward the isoelectric point as well as more rapid dissociation of ß-endorphin amyloid fibrils at lower pH indicate the contribution of ion-specific effects into dynamics of the amyloid. Several low-molecular-weight carbohydrates exhibit the same effect on ß-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, ß-endorphin amyloid fibrils obtained in the presence of heparin demonstrated distinctly different behavior, which we attributed to a dramatic change of the amyloid structure. Overall, the presented results support the hypothesis that packing of peptide hormones/neuropeptides in dense-core vesicles do not necessarily require a specialized cellular machinery.

The production of recombinant (15)N, (13)C-labelled somatostatin 14 for NMR spectroscopy.

Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer polypeptide product lowered the yield of the target peptide and complicated its purification. The side product contained the N-terminal 6His-tag together with the thioredoxin fusion partner and the specific enzymatic cleavage site-containing linker followed by an unknown peptide starting with the first 7N-terminal amino acid residues of SS14, as revealed by the Edman degradation. The combination of DNA sequence analysis, the Edman degradation, and high-resolution mass spectrometry allowed to identify the amino acid sequence of the unknown peptide. The appearance of the side product was attributed to inefficient termination of mRNA translation. The stop codon and its downstream sequence optimization allowed eliminating the side product synthesis. The optimized expression system, purification protocol, and post-translational modification procedure yielded 1.5mg of SS14 per liter of minimal medium. Nearly 99% incorporation of (13)C and (15)N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.

Fluorescence resonance energy transfer of gas-phase ions under ultra high vacuum and ambient conditions.

We report evidence for fluorescence resonance energy transfer (FRET) of gas-phase ions under ultra high vacuum conditions (10(-9) mbar) inside a mass spectrometer as well as under ambient conditions inside an electrospray plume. Two different FRET pairs based on carboxyrhodamine 6G (donor) and ATTO590 or Bodipy TR (acceptor) dyes were examined and their gas-phase optical properties were studied. Our measurements indicate a different behavior for the two FRET pairs, which can be attributed to their different conformations in the gas phase. Upon desolvation via electrospray ionization, one of the FRET pairs undergoes a conformational change that leads to disappearance of FRET. This study shows the promise of FRET to obtain a direct correlation between solution and gas-phase structures.

Mass spectrometry research at the Laboratory for Organic Chemistry, ETH Zurich.

This contribution covers the most important activities of the Zenobi research group at the Organic Chemistry Laboratory, ETH Zurich. We work in a number of interrelated areas that encompass fundamental/mechanistic research, instrument and methods development, and applications. This is illustrated with examples from the mass spectrometric study of noncovalent interactions, using both native ESI and MALDI for ionization, the investigation of the gas-phase conformation of ionized bio-macromolecules, the use of ambient mass spectrometry for rapid, on-line analyses of, for example, exhaled breath, and the use of MALDI and microarray technologies for studying metabolites with extreme sensitivity, sufficient to probe the metabolites from single cells.

Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma.

Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then re-engineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) - an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is strongly impaired. The apical annuli, therefore, represent a second type of IMC-embedded structure to the apical complex that is specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions.

DNA oligonucleotides: a model system with tunable binding strength to study monomer-dimer equilibria with electrospray ionization-mass spectrometry.

Electrospray ionization (ESI) is increasingly used to measure binding strengths, but it is not always clear whether the ESI process introduces artifacts. Here we propose a model monomer-dimer equilibrium system based on DNA oligonucleotides to systematically explore biomolecular self-association with the ESI-mass spectrometry (MS) titration method. The oligonucleotides are designed to be self-complementary and have the same chemical composition and mass, allowing for equal ionization probability, ion transmission, and detection efficiency in ESI-MS. The only difference is the binding strength, which is determined by the nucleotide sequence and can be tuned to cover a range of dissociation constant values. This experimental design allows one to focus on the impact of ESI on the chemical equilibrium and to avoid the other typical sources of variation in ESI-MS signal responses, which yields a direct comparison of samples with different binding strengths. For a set of seven model DNA oligonucleotides, the monomer-dimer binding equilibrium was probed with the ESI-MS titration method in both positive and negative ion modes. A mathematical model describing the dependence of the monomer-to-dimer peak intensity ratio on the DNA concentration was proposed and used to extract apparent Kd values and the fraction of DNA duplex that irreversibly dissociates in the gas phase. The Kd values determined via ESI-MS titration were compared to those determined in solution with isothermal titration calorimetry and equilibrium thermal denaturation methods and were found to be significantly lower. The observed discrepancy was attributed to a greater electrospray response of dimers relative to that of monomers.

A new, modular mass calibrant for high-mass MALDI-MS.

The application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of high-mass proteins requires suitable calibration standards at high m/z ratios. Several possible candidates were investigated, and concatenated polyproteins based on recombinantly expressed maltodextrin-binding protein (MBP) are shown here to be well-suited for this purpose. Introduction of two specific recognition sites into the primary sequence of the polyprotein allows for the selective cleavage of MBP3 into MBP and MBP2. Moreover, these MBP2 and MBP3 oligomers can be dimerized specifically, such that generation of MPB4 and MBP6 is possible as well. With the set of calibrants presented here, the m/z range of 40-400 kDa is covered. Since all calibrants consist of the same species and differ only in mass, the ionization efficiency is expected to be similar. However, equimolar mixtures of these proteins did not yield equal signal intensities on a detector specifically designed for detecting high-mass molecules.

Ion mobility spectrometry coupled to laser-induced fluorescence.

We report on interfacing a differential mobility analyzer (DMA) with laser-induced fluorescence (LIF) to simultaneously retrieve two-dimensional information on the electrical mobility and fluorescence spectroscopy of gas-phase ions. The fact that the separation of ions within DMA takes place in space rather than in time allows for the continuous selection of ion beams within a narrow range of mobilities that are further analyzed by LIF. Combination of DMA with LIF is simple and robust. It allows one to detect fluorescence from specified ions, including clusters, which would not survive in a mass spectrometer. Complex mixtures of fluorescent compounds can be separated by the DMA and studied by LIF. LIF is a sensitive technique and useful in the study of molecular interactions. DMA with LIF detection can be used for studies of gas-phase fluorescence of small molecules such as different dyes and their conjugates. This unique instrument combination may also provide a powerful platform for probing fluorescent proteins in the gas phase, which is of great fundamental interest for better understanding of their physical and chemical properties. In the present work, we have studied the gas-phase laser-induced fluorescence of mobility-selected rhodamine 6G ions.

Native biomolecules in the gas phase? The case of green fluorescent protein.

Green fluorescent protein (GFP) was ionized by native electrospray ionization and trapped for many seconds in high vacuum, allowing fluorescence emission to be measured as a probe of its biological function, to answer the question whether GFP exists in the native form in the gas phase or not. Although a narrow charge-state distribution, a collision cross-section very close to that expected for correctly folded GFP, and a large stability against dissociation all support a near-native gas-phase structure, no fluorescence emission was observed. The loss of the native form is attributed to the absence of residual water in the gas phase, which normally stabilizes the para-hydroxybenzylidene imidazolone chromophore of GFP.

Cryptosporidium uses multiple distinct secretory organelles to interact with and modify its host cell.

Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.

Mapping diversity in African trypanosomes using high resolution spatial proteomics.

African trypanosomes are dixenous eukaryotic parasites that impose a significant human and veterinary disease burden on sub-Saharan Africa. Diversity between species and life-cycle stages is concomitant with distinct host and tissue tropisms within this group. Here, the spatial proteomes of two African trypanosome species, Trypanosoma brucei and Trypanosoma congolense, are mapped across two life-stages. The four resulting datasets provide evidence of expression of approximately 5500 proteins per cell-type. Over 2500 proteins per cell-type are classified to specific subcellular compartments, providing four comprehensive spatial proteomes. Comparative analysis reveals key routes of parasitic adaptation to different biological niches and provides insight into the molecular basis for diversity within and between these pathogen species.

Stable endocytic structures navigate the complex pellicle of apicomplexan parasites.

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria.

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated ß-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus ß-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Quantifying protein-protein interactions within noncovalent complexes using electrospray ionization mass spectrometry.

Several electrospray-mass spectrometry (ESI-MS)-based methods are available for determining the constant of association (K(a)) between a protein and a small ligand, but current MS-based strategies are not fully adequate for measuring K(a) of protein-protein interactions accurately. We expanded the application of ESI-MS-based titration to determine the strength of noncovalent interactions between proteins, forming a complex. Taking into account relative response factors (probability of being ionized, transmitted, and detected), we determined K(a) values of an equilibrium between dimers and tetramers at three different pH values (6.8, 3.4, and 8.4). We investigated the association of the lectin concanavalin A, whose dimer-tetramer ratio in the gas phase is affected by solution concentration and by pH. To calculate the constants of association in solution, we also utilized isothermal titration calorimetry (ITC) for a comparison with MS-based titration. At pH 6.8 and pH 8.4, the K(a) values measured by MS and by ITC were in agreement. ITC results allowed us to restrain the response factor to a value close to 4. At pH 3.4, we were able to measure the K(a) only by MS, but not by ITC because of limited sensitivity of calorimetry. Our investigation illustrates the great potential MS for calculating the binding strength of protein-protein interactions within noncovalent complexes. The main advantages of MS over ITC are its sensitivity (i.e., the required amount of sample is >100 times less than the one necessary for ITC), and the possibility to obtain precise information on composition of protein complexes, their stoichiometry, their subunit interactions, and their assembly pathway. Compared to previous investigations, our study shows the strong influence of response factors on determining accurate protein-protein association constants by MS.

Aptamer-ligand recognition studied by native ion mobility-mass spectrometry.

The range of applications for aptamers, small oligonucleotide-based receptors binding to their targets with high specificity and affinity, has been steadily expanding. Our understanding of the mechanisms governing aptamer-ligand recognition and binding is however lagging, stymieing the progress in the rational design of new aptamers and optimization of the known ones. Here we demonstrate the capabilities and limitations of native ion mobility-mass spectrometry for the analysis of their higher-order structure and non-covalent interactions. A set of related cocaine-binding aptamers, displaying a range of folding properties and ligand binding affinities, was used as a case study in both positive and negative electrospray ionization modes. Using carefully controlled experimental conditions, we probed their conformational behavior and interactions with the high-affinity ligand quinine as a surrogate for cocaine. The ratios of bound and unbound aptamers in the mass spectra were used to rank them according to their apparent quinine-binding affinity, qualitatively matching the published ranking order. The arrival time differences between the free aptamer and aptamer-quinine complexes were consistent with a small ligand-induced conformational change, and found to inversely correlate with the affinity of binding. This mass spectrometry-based approach provides a fast and convenient way to study the molecular basis of aptamer-ligand recognition.

A Prioritized and Validated Resource of Mitochondrial Proteins in Plasmodium Identifies Unique Biology.

Plasmodium species have a single mitochondrion that is essential for their survival and has been successfully targeted by antimalarial drugs. Most mitochondrial proteins are imported into this organelle, and our picture of the Plasmodium mitochondrial proteome remains incomplete. Many data sources contain information about mitochondrial localization, including proteome and gene expression profiles, orthology to mitochondrial proteins from other species, coevolutionary relationships, and amino acid sequences, each with different coverage and reliability. To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight data sets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 445 proteins with a sensitivity of 87% and a specificity of 97%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. Mitochondrial localization of seven Plasmodium berghei orthologs was confirmed by epitope labeling and colocalization with a mitochondrial marker protein. One of these belongs to a newly identified apicomplexan mitochondrial protein family that in P. falciparum has four members. With the experimentally validated mitochondrial proteins and the complete ranked P. falciparum proteome, which we have named PlasmoMitoCarta, we present a resource to study unique proteins of Plasmodium mitochondria. IMPORTANCE The unique biology and medical relevance of the mitochondrion of the malaria parasite Plasmodium falciparum have made it the subject of many studies. However, we actually do not have a comprehensive assessment of which proteins reside in this organelle. Many omics data are available that are predictive of mitochondrial localization, such as proteomics data and expression data. Individual data sets are, however, rarely complete and can provide conflicting evidence. We integrated a wide variety of available omics data in a manner that exploits the relative strengths of the data sets. Our analysis gave a predictive score for the mitochondrial localization to each nuclear encoded P. falciparum protein and identified 445 likely mitochondrial proteins. We experimentally validated the mitochondrial localization of seven of the new mitochondrial proteins, confirming the quality of the complete list. These include proteins that have not been observed mitochondria before, adding unique mitochondrial functions to P. falciparum.

Rhodamines in the gas phase: cations, neutrals, anions, and adducts with metal cations.

Optical spectroscopy of biological molecules in the gas phase has recently gained considerable attention, being able to provide complementary structural information in the absence of native matrix. Biomolecules can change their properties when brought into the gas phase, and so can chromophores associated with them. Understanding the photophysics of chromophore labels is central for the correct interpretation of experimental data. In this report, the structure and the optical properties of Rhodamine 19 (R19) in the gas phase were examined by a combination of Fourier-transform ion cyclotron resonance mass spectrometry and visible-light laser spectroscopy. While R19 in solution is found either in neutral (R19(n)) or protonated (R19+H(+)) forms, other structures can be generated in the gas phase, such as anions (R19-H(-)) and adducts with metal cations (R19+M(+)). Experimental evidence for the lactone structure of neutral gas-phase R19 is presented for the first time. The different properties of gas-phase compared to solution-phase R19 are discussed in view of structural analysis of labeled gas-phase biological molecules by optical spectroscopy.