Laser‐induced liquid bead ion desorption‐MS of protein complexes from blue‐native gels,a sensitive top‐down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.     

Reconstitution of the multiprotein complex of pp60src, hsp90, and p50 in a cell-free system.

A rabbit reticulocyte lysate system that has been used to reconstitute functional complexes between steroid receptors and the 90-kDa heat shock protein (hsp90) has been used here to form complexes between the pp60src tyrosine kinase and hsp90. Reticulocyte lysate forms complexes between hsp90 and a temperature-sensitive mutant of Rous sarcoma virus pp60v-src, which is normally present in cytosol virtually entirely in the multiprotein complex form. In addition, hsp90 in the lysate complexes with wild-type pp60v-src, of which only a small portion is normally recovered in cytosol in the native multiprotein complex, and with the cellular homolog, pp60c-src, which has never been recovered in cytosol in the form of a native multiprotein complex with hsp90. Moreover, the reticulocyte lysate-reconstituted complex also contains the 50-kDa phosphoprotein component of the native pp60v-src multiprotein complex. The native and reconstituted pp60src-hsp90 complexes have similar thermal stability and, like steroid receptor heterocomplexes, they are stabilized by molybdate. As previously shown with reticulocyte lysate-reconstituted steroid receptor heteroprotein complexes, the reconstituted pp60src multiprotein complex contains hsp70, which is a major candidate for providing the protein unfoldase activity required for hsp90 association.     

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (¹⁴N/¹⁵N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.     

Chromatin-modifying and -remodeling complexes.

Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure.     

Subunit mass fingerprinting of mitochondrial complex I

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.     

Identification and characterization of protein subcomplexes in yeast

Protein complexes are major components of cellular organization. Based on large-scale protein complex data, we present the first statistical procedure to find insightful substructures in protein complexes: we identify protein subcomplexes (SCs), i.e., multiprotein assemblies residing in different protein complexes. Four protein complex datasets with different origins and variable reliability are separately analyzed. Our method identifies well-characterized protein assemblies with known functions, thereby confirming the utility of the procedure. In addition, we also identify hitherto unknown functional entities consisting of either functionally unknown proteins or proteins with different functional annotation. We show that SCs represent more reliable protein assemblies than the original complexes. Finally, we demonstrate unique properties of subcomplex proteins that underline the distinct roles of SCs: (i) SCs are functionally and spatially more homogeneous than complete protein complexes (this fact is utilized to predict functional roles and subcellular localizations for so far unannotated proteins); (ii) the abundance of subcomplex proteins is less variable than the abundance of other proteins; (iii) SCs are enriched with essential and synthetic lethal proteins; and (iv) mutations in SC-proteins have higher fitness effects than mutations in other proteins.     

Tethered function assays using 3' untranslated regions

Proteins that regulate mRNA metabolism are often bipartite: an RNA binding activity confers substrate specificity, and a "functional" domain elicits the biological outcome. In some cases, these two activities reside on different polypeptides that form a complex on the mRNA. Regardless, experimental separation of RNA binding from function facilitates analysis of both properties, liberating each from the constraints of the other. "Tethered function" assays bring a protein to a reporter RNA through a designed RNA-protein interaction. The function of the tethered protein-whether that be stability, translation, localization, or transport, or otherwise-is then assessed. We refer to this approach as a "tethered function" assay, since it can be examined. The approach does not require knowledge of the natural RNA binding sites, or of the composition of the naturally occurring protein complexes. It can be useful in dissecting how proteins that act on RNAs work, and in identifying active components of multiprotein complexes. RNA-binding proteins previously have been linked to foreign RNA-binding specificities, for a wide variety of purposes. We emphasize here the particular value of tethering to the 3' untranslated region of eukaryotic mRNAs, and the opportunities it presents for the analysis of how those mRNAs are regulated. We discuss experimental design, as well as published and potential applications.     

Analysis of the Plasmodium falciparum proteasome using Blue Native PAGE and label-free quantitative mass spectrometry

Detailed knowledge of the composition of protein complexes is crucial for the understanding of their structure and function; however, appropriate techniques for compositional analyses of complexes largely rely on elaborate tagging, immunoprecipitation, cross-linking and purification strategies. The proteasome is a prototypical protein complex and therefore an excellent model to assess new methods for protein complex characterisation. Here we evaluated the applicability of Blue Native (BN) PAGE in combination with label-free protein quantification and protein correlation profiling (PCP) for the investigation of proteasome complexes directly from biological samples. Using the purified human 20S proteasome we showed that the approach can accurately detect members of a complex by clustering their gel migration profiles. We applied the approach to address proteasome composition in the schizont stage of the malaria parasite Plasmodium falciparum. The analysis, performed in the background of the whole protein extract, revealed that all subunits comigrated and formed a tight cluster with a single maximum, demonstrating presence of a single form of the 20S proteasome. This study shows that BN PAGE in combination with label-free quantification and PCP is applicable to the analysis of multiprotein complexes directly from complex protein mixtures.     

The integration of signaling by multiprotein complexes containing Raf kinases

In vivo, eukaryotic cells are subjected simultaneously to a broad array of signals ranging from mitogens and inflammatory inputs to environmental stresses and developmental cues. The combinatorial nature of cellular signaling necessitates that a cell integrate its signal transduction pathways so as to implement rapidly and efficiently an appropriate suite of responses. Emerging evidence indicates that, over the course of evolution, cells have developed multiprotein signaling complexes, or "signalosomes" that mediate the coordinate regulation of different signaling pathways. Such molecular signal integration contrasts with the classical notion of signaling complexes assembled by scaffold proteins-entities that function to segregate specific pathways from one another. This review will focus on two signal integrating multiprotein complexes that involve Raf family kinases: the MLK3-B-Raf-Raf-1 complex and the Raf-1-Mst-2 complex.     

Knowledge-based real-space explorations for low-resolution structure determination

The accurate and effective interpretation of low-resolution data in X-ray crystallography is becoming increasingly important as structural initiatives turn toward large multiprotein complexes. Substantial challenges remain due to the poor information content and ambiguity in the interpretation of electron density maps at low resolution. Here, we describe a semiautomated procedure that employs a restraint-based conformational search algorithm, RAPPER, to produce a starting model for the structure determination of ligase interacting factor 1 in complex with a fragment of DNA ligase IV at low resolution. The combined use of experimental data and a priori knowledge of protein structure enabled us not only to generate an all-atom model but also to reaffirm the inferred sequence registry. This approach provides a means to extract quickly from experimental data useful information that would otherwise be discarded and to take into account the uncertainty in the interpretation--an overriding issue for low-resolution data.     

New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes

NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties. Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA. The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts. These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes. Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented.     

Binding specificity of multiprotein signaling complexes is determined by both cooperative interactions and affinity preferences

The generation of multiprotein complexes at receptors and adapter proteins is crucial for the activation of intracellular signaling pathways. In this study, we used multiple biochemical and biophysical methods to examine the binding properties of several SH2 and SH3 domain-containing signaling proteins as they interact with the adapter protein linker for activation of T-cells (LAT) to form multiprotein complexes. We observed that the binding specificity of these proteins for various LAT tyrosines appears to be constrained both by the affinity of binding and by cooperative protein-protein interactions. These studies provide quantitative information on how different binding parameters can determine in vivo binding site specificity observed for multiprotein signaling complexes.     

Scaffold Protein Connector Enhancer of Kinase Suppressor of Ras Isoform 3 (CNK3) Coordinates Assembly of a Multiprotein Epithelial Sodium Channel (ENaC)-regulatory Complex

Hormone regulation of ion transport in the kidney tubules is essential for fluid and electrolyte homeostasis in vertebrates. A large body of evidence has suggested that transporters and channels exist in multiprotein regulatory complexes; however, relatively little is known about the composition of these complexes or their assembly. The epithelial sodium channel (ENaC) in particular is tightly regulated by the salt-regulatory hormone aldosterone, which acts at least in part by increasing expression of the serine-threonine kinase SGK1. Here we show that aldosterone induces the formation of a 1.0–1.2-MDa plasma membrane complex, which includes ENaC, SGK1, and the ENaC inhibitor Nedd4-2, a key target of SGK1. We further show that this complex contains the PDZ domain-containing protein connector enhancer of kinase suppressor of Ras isoform 3 (CNK3). CNK3 physically interacts with ENaC, Nedd4-2, and SGK1; enhances the interactions among them; and stimulates ENaC function in a PDZ domain-dependent, aldosterone-induced manner. These results strongly suggest that CNK3 is a molecular scaffold, which coordinates the assembly of a multiprotein ENaC-regulatory complex and hence plays a central role in Na⁺ homeostasis.