Micropreparative fractionation of the complexome by blue native continuous elution electrophoresis

The large‐scale analysis of protein complexes is an emerging challenge in the field of proteomics. Currently, there are few methods available for the fractionation of protein complexes that are compatible with downstream proteomic techniques. Here, we describe the technique of blue native continuous elution electrophoresis (BN‐CEE). It combines the features of blue native PAGE (BN‐PAGE) and continuous elution electrophoresis (CEE), generating liquid‐phase fractions of protein complexes of up to 800 kDa. The resulting complexes can be further analysed by BN‐PAGE, by SDS‐PAGE and/or by MS. This can help define the constituent proteins of many complexes and their stoichiometry. As BN‐CEE is also micropreparative, with a capacity to separate milligram quantities of protein complexes, it will assist the study of proteins of lower abundance. In this regard, the acrylamide concentration and elution rate during separation can be controlled to help ‘zoom in’ on particular high mass regions and thus complexes of interest. We illustrate the utility of the technique in the analysis of Saccharomyces cerevisiae cellular lysate.     

Characterization of the control catabolite protein of gluconeogenic genes repressor by fluorescence cross-correlation spectroscopy and other biophysical approaches

Determination of the physical parameters underlying protein-DNA interactions is crucial for understanding the regulation of gene expression. In particular, knowledge of the stoichiometry of the complexes is a prerequisite to determining their energetics and functional molecular mechanisms. However, the experimental determination of protein-DNA complex stoichiometries remains challenging. We used fluorescence cross-correlation spectroscopy (FCCS) to investigate the interactions of the control catabolite protein of gluconeogenic genes, a key metabolic regulator in Gram-positive bacteria, with two oligonucleotides derived from its target operator sequences, gapB and pckA. According to our FCCS experiments, the stoichiometry of binding is twofold larger for the pckA target than for gapB. Correcting the FCCS data for protein self-association indicated that control catabolite protein of gluconeogenic genes forms dimeric complexes on the gapB target and tetrameric complexes on the pckA target. Analytical ultracentrifugation coupled with fluorescence anisotropy and hydrodynamic modeling allowed unambiguous confirmation of this result. The use of multiple complementary techniques to characterize these complexes should be employed wherever possible. However, there are cases in which analytical ultracentrifugation is precluded, due to protein stability, solubility, or availability, or, more obviously, when the studies are carried out in live cells. If information concerning the self-association of the protein is available, FCCS can be used for the direct and simultaneous determination of the affinity, cooperativity, and stoichiometry of protein-DNA complexes in a concentration range and conditions relevant to the regulation of these interactions.     

Size-Exclusion Chromatography with On-Line Light-Scattering, Absorbance, and Refractive Index Detectors for Studying Proteins and Their Interactions

Techniques of using size-exclusion chromatography (SEC) with on-line light-scattering, uv absorbance, and refractive index detectors to characterize the polypeptide molecular weights of simple proteins or glycoproteins or to determine the stoichiometry of protein complexes are described. Two unique advantages of this approach over conventional SEC are that the molecular weight measurement is independent of elution position and can exclude the contributions from carbohydrates. When a protein or complex contains no carbohydrates, a two-detector method, i.e., light scattering combined with refractive index, can be used to calculate the molecular weight. When a protein contains carbohydrates, a three-detector method is used to calculate the molecular weight of polypeptide alone. Finally, a self-consistent three-detector method is used to determine the stoichiometry of a protein complex containing carbohydrates. Example applications for all these methodologies are described.     

Analyzing chromatin remodeling complexes using shotgun proteomics and normalized spectral abundance factors

Mass spectrometry-based approaches are commonly used to identify proteins from multiprotein complexes, typically with the goal of identifying new complex members or identifying post-translational modifications. However, with the recent demonstration that spectral counting is a powerful quantitative proteomic approach, the analysis of multiprotein complexes by mass spectrometry can be reconsidered in certain cases. Using the chromatography-based approach named multidimensional protein identification technology, multiprotein complexes may be analyzed quantitatively using the normalized spectral abundance factor that allows comparison of multiple independent analyses of samples. This study describes an approach to visualize multiprotein complex datasets that provides structure function information that is superior to tabular lists of data. In this method review, we describe a reanalysis of the Rpd3/Sin3 small and large histone deacetylase complexes previously described in a tabular form to demonstrate the normalized spectral abundance factor approach.     

Dependency and independency among fish density and electivity indices in a stream fish assemblage

Habitat preferences can be identified from population density and electivity indices, which can be derived from either abundance or occurrence data. This study examined the preference detectability and independency of density and electivity indices from different data sources -- abundance and occurrence. Eight scenarios comprising four fish species along two environmental gradients (flow velocity and water depth) in an undisturbed mountain stream were used in this study. Fish density and seven electivity indices were calculated from abundance and occurrence data for the eight scenarios. The correlation analyses illustrated that abundance electivity indices were positively correlated with density in all eight scenarios while occurrence electivity indices were positively correlated with density in only three scenarios. Electivity indices were positively correlated with each other within the same data quality (abundance or occurrence) but not between. The Chesson’s L index was less correlated with other indices and should be used cautiously. Natural historical traits, such as social interactions among organisms, may affect the preference performance of density and electivity indices. In conclusion, density is a good preference indicator. Abundance electivity indices can represent density while occurrence electivity density should be used cautiously for fast assessment. Three electivity indices, Ivlev’s E and E′ and Jacobs’ D, were recommended for their ease of understanding.     

Is there a correlation between abundance and environmental suitability derived from ecological niche modelling? A meta‐analysis

It is thought that species abundance is correlated with environmental suitability and that environmental variables, scale, and type of model fitting can confound this relationship. We performed a meta‐analysis to 1) test whether species abundance is positively correlated with environmental suitability derived from correlative ecological niche models (ENM), 2) test whether studies encompassing large areas within a species range (> 50%) exhibited higher AS correlations than studies encompassing small areas within a species range (< 50%), 3) assess which modelling method provided higher AS correlation, and 4) compare strength of the AS relationship between studies using only climatic variables and those that used both climatic and other environmental variables to derive suitability. We used correlation coefficients to measure the relationship between abundance and environmental suitability derived from ENM. Each correlation coefficient was considered an effect size in a random‐effects multivariate meta‐analysis. In all cases we found a significantly positive relationship between abundance and suitability. This relationship was consistent regardless of scale of study, ENM method, or set of variables used to derive suitability. There was no difference in strength of correlation between studies focusing on large or small areas within a species’ range or among ENM methods. Studies using other variables in combination with climate exhibited higher AS correlations than studies using only climatic variables. We conclude that occurrence data can be a reasonable proxy for abundance, especially for vertebrates, and the use of local variables increases the strength of the AS relationship. Use of ENMs can significantly decrease survey costs and allow the study of large‐scale abundance patterns using less information. Including only climatic variables in ENM may confound the relationship between abundance and suitability when compared to studies including variables taken locally. However, modelers and conservationists must be aware that high environmental suitability does not always indicate high abundance.     

Cu(I) binding to the Schizosaccharomyces pombe gamma-glutamyl peptides varying in chain lengths

The metal-gamma-glutamyl peptide complex of Schizosaccharomyces pombe is an oligomer of peptides of the general structure (gamma-Glu-Cys)n-Gly with n defining the number of dipeptide repeats. The complexes induced with either cadmium or copper salts are heterogeneous with respect to the number of repeat units or n. Peptides isolated from two preparations of the Cd-gamma-Glu complex by reverse-phase HPLC at low pH were of an n range of 2 to 6 with n3 and n4 peptides being predominant. In addition to peptides of the mentioned structure, peptides of n3 and n4 without the terminal Gly were isolated. These n3 and n4 desGly peptides were present in an abundance of about 10-20% of the concentration of the parent peptide. Peptides of unique n were studied in Cu(I) reconstitution experiments in an attempt to understand the significance of the peptide length heterogeneity in the oligomeric metal-thiolate cluster. Cu-gamma-Glu complexes were formed with each peptide as determined by the characteristic 260-nm shoulder in the ultraviolet absorption spectrum and luminescence indicative of Cu(I)-thiolate coordination in a solvent-inaccessible environment. Cluster formation also occurs with desGly peptides, so the carboxyl-terminal Gly is not critical for cluster formation. Maximal Cu binding stoichiometry with n3 and n4 peptides was markedly less than the maximal Cu(I) stoichiometry of a peptide mixture or the native complex. Cu ions in complexes formed with unique n peptides were more reactive with bathocuproine than Cu ions in complexes with a peptide n mixture. The results suggest that metal-peptide complexes consisting of peptides differing in n probably exist and not all metal-peptide complexes have the same n peptide constituents.     

Subunit architecture of multimeric complexes isolated directly from cells

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.     

Distinct Cellular Assembly Stoichiometry of Polycomb Complexes on Chromatin Revealed by Single-molecule Chromatin Immunoprecipitation Imaging

Epigenetic complexes play an essential role in regulating chromatin structure, but information about their assembly stoichiometry on chromatin within cells is poorly understood. The cellular assembly stoichiometry is critical for appreciating the initiation, propagation, and maintenance of epigenetic inheritance during normal development and in cancer. By combining genetic engineering, chromatin biochemistry, and single-molecule fluorescence imaging, we developed a novel and sensitive approach termed single-molecule chromatin immunoprecipitation imaging (Sm-ChIPi) to enable investigation of the cellular assembly stoichiometry of epigenetic complexes on chromatin. Sm-ChIPi was validated by using chromatin complexes with known stoichiometry. The stoichiometry of subunits within a polycomb complex and the assembly stoichiometry of polycomb complexes on chromatin have been extensively studied but reached divergent views. Moreover, the cellular assembly stoichiometry of polycomb complexes on chromatin remains unexplored. Using Sm-ChIPi, we demonstrated that within mouse embryonic stem cells, one polycomb repressive complex (PRC) 1 associates with multiple nucleosomes, whereas two PRC2s can bind to a single nucleosome. Furthermore, we obtained direct physical evidence that the nucleoplasmic PRC1 is monomeric, whereas PRC2 can dimerize in the nucleoplasm. We showed that ES cell differentiation induces selective alteration of the assembly stoichiometry of Cbx2 on chromatin but not other PRC1 components. We additionally showed that the PRC2-mediated trimethylation of H3K27 is not required for the assembly stoichiometry of PRC1 on chromatin. Thus, these findings uncover that PRC1 and PRC2 employ distinct mechanisms to assemble on chromatin, and the novel Sm-ChIPi technique could provide single-molecule insight into other epigenetic complexes.     

Crystal structures, antioxidation and DNA binding properties of Yb(III) complexes with Schiff-base ligands derived from 8-hydroxyquinoline-2-carbaldehyde and four aroylhydrazines

X-ray crystal and other structural analyses indicate that Yb(III) and all four newly synthesized ligands can form a binuclear Yb(III) complex with a 1:1 metal to ligand stoichiometry by octacoordination at the Yb(III) center. Investigations of DNA binding properties show that all the ligands and Yb(III) complexes can bind to Calf thymus DNA through intercalations with the binding constants at the order of magnitude 10⁵–10⁷ M⁻¹, but Yb(III) complexes present stronger affinities to DNA than ligands. All the ligands and Yb(III) complexes may be used as potential anticancer drugs. Investigations of antioxidation properties show that all the ligands and Yb(III) complexes have strong scavenging effects for hydroxyl radicals and superoxide radicals but Yb(III) complexes show stronger scavenging effects for hydroxyl radicals than ligands. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of selected reaction monitoring data for label-free quantification of protein modification stoichiometry

Quantification of protein and PTM abundance in biological samples is an important component of proteomic studies. Label-free methods for quantification using MS are attractive because they are simple to implement and applicable to any experimental system. We demonstrate that PTM stoichiometry can be accurately measured using label-free quantification and selected reaction monitoring. Use of selected reaction monitoring is advantageous with complex biological samples and we show this approach can be used to quantify multiple PTMs independently on a single peptide.     

Autoregressive modeling of analytical sensor data can yield classifiers in the predictor coefficient parameter space

SUMMARY: The analysis of chromatographic data resulting from complex chemical mixtures is challenging. Components may co-elute, causing their signals to overlap. An algorithm that will increase the signal-to-noise ratio so compounds present in low abundance can be better distinguished from noise is useful in this type of analysis. The autoregressive (AR) filter offers the advantage of smoothing chromatograms to increase this ratio, while also offering data compression and increased resolution. Furthermore, this filter can be useful for classification, as the roots of the predictor coefficient vectors represent features present in the data and can therefore be used for pattern recognition. In this paper, we present a novel method for applying AR filtering to chromatogram data. We show that the AR filter outperforms the Savitzky-Golay filter for smoothing noise while retaining important information within chromatograms, and also that AR correlation coefficients have the potential to be used to classify chromatogram data into groups. CONTACT: cdavis@draper.com.     

Determination of protein complex stoichiometry through multisignal sedimentation velocity experiments

Determination of the stoichiometry of macromolecular assemblies is fundamental to an understanding of how they function. Many different biophysical methodologies may be used to determine stoichiometry. In the past, both sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation have been employed to determine component stoichiometries. Recently, a method of globally analyzing multisignal sedimentation velocity data was introduced by Schuck and coworkers. This global analysis removes some of the experimental inconveniences and inaccuracies that could occur in the previously used strategies. This method uses spectral differences between the macromolecular components to decompose the well-known c(s) distribution into component distributions c_k(s); that is, each component k has its own c_k(s) distribution. Integration of these distributions allows the calculation of the populations of each component in cosedimenting complexes, yielding their stoichiometry. In our laboratories, we have used this method extensively to determine the component stoichiometries of several protein-protein complexes involved in cytoskeletal remodeling, sugar metabolism, and host-pathogen interactions. The overall method is described in detail in this work, as are experimental examples and caveats.     

Identification of key microhabitats for fish assemblages in tropical Brazilian savanna streams

The habitat use by stream fish can provide an understanding of this important component of species niches, and the changes in the availability of certain structures of instream habitats have a strong influence on the occurrence and abundance of fish species. In this study, we identified the interaction networks between fish and microhabitats in 19 streams of three watersheds from the Brazilian savanna, known as the “Cerrado”. We also investigated whether the connectance between fish species and microhabitats depends on either the species abundance or availability of microhabitats and verified whether the use of stream microhabitats vary among watersheds. The data for the fish were obtained using standardized fish collections and underwater (snorkeling) observations to record the association of fish species with 11 stream microhabitats. We used the incidence data to calculate the connectance of species with different microhabitats, a Spearman correlation analysis to test the dependence of the connectance to the species abundance and microhabitat availability, and an Analysis of Similarity to test whether the use of stream microhabitats by fish can vary among watersheds. In all of the watersheds, the tetras of the family Characidae used the largest number of microhabitats. The most‐used microhabitats by the fish watersheds were the lateral surface, column surface, and unconsolidated substrate, which were differentially used by the fish in each watershed, indicating the variable importance of microhabitats. Despite this observation, the fish occurred more frequently in the lateral microhabitats. When the data for all of the streams were combined, the species connectance was correlated with the species abundance. We also discuss the consequences of the elimination of certain bottom structures in the context of siltation, which is the main issue in the studied region. The results of our study allowed the detection of key microhabitat structures and can be used for the aquatic ecosystems restoration. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Stoichiometry of protein complexes in plant photosynthetic membranes

Hetero-oligomeric membrane protein complexes form the electron transport chain (ETC) of oxygenic photosynthesis. The ETC complexes undertake the light-driven vectorial electron and proton transport reactions, which generate energy-rich ATP and electron-rich NADPH molecules for carbon fixation. The rate of photosynthetic electron transport depends on the availability of photons and the relative abundance of electron transport complexes. The relative abundance of the two photosystems, critical for the quantum efficiency of photosynthesis in changing light quality conditions, has been determined successfully by optical methods. Due to the lack of spectroscopic signatures, however, relatively little is known about the stoichiometry of other non-photosystem complexes in plant photosynthetic membrane. Here we determine the ratios of all major thylakoid-bound ETC complexes in Arabidopsis by a label-free quantitative mass spectrometry technique. The calculated stoichiometries are consistent with known subunit composition of complexes and current estimates of photosystem and cytochrome b₆f concentrations. The implications of these stoichiometries for photosynthetic light harvesting and the partitioning of electrons between the linear and cyclic electron transport pathways of photosynthesis are discussed.     

Nutrients and Other Abiotic Factors Affecting Bacterial Communities in an Ohio River (USA)

Nitrogen and phosphorus additions from anthropogenic sources can alter the nutrient pool of aquatic systems, both through increased nutrient concentrations and changes in stoichiometry. Because bacteria are important in nutrient cycling and aquatic food webs, information about how nutrients affect bacterial communities enhances our understanding of how changes in nutrient concentrations and stoichiometry potentially affect aquatic ecosystems as a whole. In this study, bacterial communities were examined in biofilms from cobbles collected across seasons at three sites along the Mahoning River (Ohio) with differing levels of inorganic nutrient inputs. Members of the alpha-, beta-, and gamma-proteobacteria, the Cytophaga–Flavobacteria cluster, and the Domain Bacteria were enumerated using fluorescent in situ hybridization. Detrended canonical correspondence analysis (DCCA) revealed that stoichiometric ratios, especially the dissolved inorganic nitrogen (DIN):soluble reactive phosphorus (SRP) molar ratio (NO₂/NO₃ + NH₄:soluble reactive phosphorus), were correlated with abundance of the various bacterial taxa. However, the patterns were complicated by correlations with single nutrient concentrations and seasonal changes in temperature. Seasonal cycles appeared to play an important role in structuring the community, as there were distinct winter communities and temperature was negatively correlated with abundance of both alpha-proteobacteria and Cytophaga–Flavobacteria. However, nutrients and stoichiometry also appeared to affect the community. Numbers of cells hybridizing the Domain Bacteria probe were correlated with the DOC:DIN ratio, the beta-proteobacteria had a negative correlation with soluble reactive phosphorus concentrations and a positive correlation with the DIN:SRP ratio, and the Cytophaga–Flavobacteria had a significant negative partial correlation with the DIN:SRP ratio. Abundances of the alpha- or gamma-proteobacteria were not directly correlated to nutrient concentrations or stoichiometry. It appears that nutrient stoichiometry may be an important factor structuring bacterial communities; however, it is one of many factors, such as temperature, that are interlinked and must be considered together when studying environmental bacteria.     

Inferring subunit stoichiometry from single molecule photobleaching

Single molecule photobleaching is a powerful tool for determining the stoichiometry of protein complexes. By attaching fluorophores to proteins of interest, the number of associated subunits in a complex can be deduced by imaging single molecules and counting fluorophore photobleaching steps. Because some bleaching steps might be unobserved, the ensemble of steps will be binomially distributed. In this work, it is shown that inferring the true composition of a complex from such data is nontrivial because binomially distributed observations present an ill-posed inference problem. That is, a unique and optimal estimate of the relevant parameters cannot be extracted from the observations. Because of this, a method has not been firmly established to quantify confidence when using this technique. This paper presents a general inference model for interpreting such data and provides methods for accurately estimating parameter confidence. The formalization and methods presented here provide a rigorous analytical basis for this pervasive experimental tool.     

Pentacopper(II) 12-metallacrown-4 complexes with alpha- and beta-aminohydroxamic acids in aqueous solution: a reinvestigation

A reinvestigation of the equilibria of (S)-alpha-alaninehydroxamic acid (alpha-Alaha) and (R)-aspartic-beta-hydroxamic acid (Asp-beta-ha) with copper(II) was performed in aqueous solution in order to clarify some contradictory literature reports regarding the stoichiometry of the polynuclear complexes formed. beta-Alaninehydroxamic acid (beta-Alaha, HL), for which the formation of a planar 12-metallacrown-4, [Cu(5)L(4)H(-4)](2+), was already reported, was also re-examined for comparison. Among the different techniques used (potentiometry, absorption spectrophotometry, spectropolarimetry and electrospray ionization mass spectrometry), ES data allowed to define unambiguously that all these three ligands form the same pentanuclear species. Therefore it can be concluded that in aqueous solution the hydroxamates of both alpha- and beta-amino acids form 12-metallacrown-4 complexes, and that the formers are less stable.