Summary Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to highly specialized cell compartments and structures. These adaptations drive their recognition, nondestructive penetration, and elaborate reengineering of the host’s cells to promote their growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty. Consequently, half of apicomplexan proteins are unique and uncharacterized. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii . This provides unprecedented comprehensive molecular definition of these unicellular eukaryotes and their specialized compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

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.

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.

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to novel, highly specialized cell compartments and structures. These adaptations drive their recognition and nondestructive penetration of host's cells and the elaborate reengineering of these cells to promote growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty that defines these new cell organizations and their functions. Consequently, half of apicomplexan proteins are unique and uncharacterized, and these cells are, therefore, very poorly understood. Here, we determine the steadystate subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition to these cells and their novel compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

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 β-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 β-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.

Electrospray ionization mass spectrometry (ESI-MS) is a powerful analytical method to study biomolecules and noncovalent complexes. The prerequisite for their intact observation is soft ionization. In ESI, the internal energy of ions is primarily influenced by collisional activation in the source. The survival yield method is frequently used to probe the energy deposition in ions during the electrospray process. In the present work, we investigate the fragmentation pathways of para-substituted benzylpyridinium ions, the most widely used "thermometer ions" in the survival yield method. In addition to the C-N bond cleavage, alternative fragmentation channels were found for the compounds studied. We consider these pathways to result from intramolecular rearrangements. The effect of these additional fragments on the accuracy of the internal energy calibration is estimated for both collision-cell and in-source collision-induced dissociation (CID). Altogether, results presented suggest that a correction of the energy scale is necessary for the method based on benzylpyridinium ions to precisely quantify ion internal energies. (J Am Soc Mass Spectrom 2010, 21, 172-177) © 2010 American Society for Mass Spectrometry E lectrospray ionization mass spectrometry (ESI-MS) [1] is widely used to characterize various species, from small organic compounds [2] to large supramolecular assemblies of biopolymers [3]. In the ESI source, small charged droplets containing dissolved analyte are produced at atmospheric pressure. As the droplets migrate along a voltage gradient to the low-pressure region of the mass spectrometer, solvent evaporates, releasing unsolvated ions. Ions produced in the ion source undergo collisions with ambient gas molecules and thereby accumulate internal energy. Such collisional activation often results in fragmentation and/or rearrangement of ions [4,5]. The effect is more pronounced for weak interactions, such as those involved in noncovalent complexes, which are widely studied by ESI-MS [5]. In addition, for instruments with restricted MS/MS capabilities, so-called in-source CID is frequently the only way to obtain structural information on the parent ions. In-source CID strongly depends on instrument parameter settings and experimental conditions, thus resulting in poor reproducibility of the MS/MS spectra [5]. Internal energy deposition in ions generally affects the mass spectra. It is therefore important to control the ion internal energy for many applications, such as optimizing the molecular ion abundance, structure determination, differentiation of isomers, MS n experiments, and in the study of non-ovalent complexes [5].The survival yield method was introduced to calibrate the internal energy distribution of ions after collisional activation [6,7]. In this method, compounds with a simple and well known dissociation pattern, so-called thermometer ions, are used to probe the energy uptake due to the activation process. The survival yield is the ratio of the parent ion intensity to the sum of parent and f...

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 β-endorphin amyloids in vitro. We found that β-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 β-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 β-endorphin aggregation as phosphate. Moreover, no structural difference was detected between the phosphate- and carbohydrate-induced fibrils by solid-state NMR. In contrast, β-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.

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 K = 69.7 μM and K = 5.3 μM for ABA (two binding sites); K = 22.04 μM for LABA; and K = 8.5 μM for CBA.