Thylakoid membrane at altered metabolic state: challenging the forgotten realms of the proteome

Analysis of the membrane integral proteome is mainly dependent on the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique capable of efficient membrane protein separation, so far mainly applied to the mitochondrial oxidative phosphorylation machinery. Applying BN-PAGE to the thylakoid membranes after mild solubilization with digitonin we succeeded in displaying the response of the green algae Chlamydomonas reinhardtii to altered culture conditions. In addition, by peptide mass fingerprinting and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) extremely hydrophobic subunits of the photosystem complexes with 5-11 transmembrane helices were identified, which could not be accessed by in-gel digestion in previous studies.     

Two-dimensional Blue Native/sodium dodecyl sulfate gel electrophoresis for analysis of multimeric proteins in platelets

Two-dimensional (2-D) Blue Native/SDS gel electrophoresis combines a first-dimensional separation of monomeric and multimeric proteins in their native state with a second denaturing dimension. These high-resolution 2-D gels aim at identifying multiprotein complexes with respect to their subunit composition. We applied this method for the first time to analyze two human platelet subproteomes: the cytosolic and the microsomal membrane protein fraction. Solubilization of platelet membrane proteins was achieved with the nondenaturing detergent n-dodecyl-beta-D-maltoside. To validate native solubilization conditions, we demonstrated the correct assembly of the Na,K-ATPase, a functional multimeric transmembrane protein, when expressed in Xenopus oocytes. We identified 63 platelet proteins after in-gel tryptic digestion of 58 selected protein spots and liquid chromatography-coupled tandem mass spectrometry. Nine proteins were detected for the first time in platelets by a proteomic approach. We also show that this technology efficiently resolves several known membrane and cytosolic multiprotein complexes. Blue Native/SDS gel electrophoresis is thus a valuable procedure to analyze specific platelet subproteomes, like the membrane(-bound) protein fraction, by mass spectrometry and immunoblotting and could be relevant for the study of protein-protein interactions generated following platelet activation.     

Efficient and non-denaturing membrane solubilization combined with enrichment of membrane protein complexes by detergent/polymer aqueous two-phase partitioning for proteome analysis

It is of central interest in membrane proteomics to establish methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. We have investigated the quantitative and qualitative solubilization efficiency of five commercially available detergents using mitochondria from the yeast Saccharomyces cerevisiae as model system. Combining the zwitterionic detergent Zwittergent 3-10 and the non-ionic detergent Triton X-114 resulted in a complementary solubilization of proteins, which was similar to that of the anionic detergent sodium dodecyl sulfate (SDS). The subsequent removal of soluble proteins by detergent/polymer two-phase system partitioning was further enhanced by addition of SDS and increasing pH. A large number of both integral and peripheral membrane protein subunits from mitochondrial membrane protein complexes were identified in the detergent phase. We suggest that the optimized solubilization protocol in combination with detergent/polymer two-phase partitioning is a mild and efficient method for initial enrichment of membrane proteins and membrane protein complexes in proteomic studies.     

Two-dimensional electrophoresis of membrane proteins

One third of all genes of various organisms encode membrane proteins, emphasizing their crucial cellular role. However, due to their high hydrophobicity, membrane proteins demonstrate low solubility and a high tendency for aggregation. Indeed, conventional two-dimensional gel electrophoresis (2-DE), a powerful electrophoretic method for the separation of complex protein samples that applies isoelectric focusing (IEF) in the first dimension and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, has a strong bias against membrane proteins. This review describes two-dimensional electrophoretic techniques that can be used to separate membrane proteins. Alternative methods for performing conventional 2-DE are highlighted; these involve replacing the IEF with electrophoresis using cationic detergents, namely 16-benzyldimethyl-n-hexadecylammonium chloride (16-BAC) and cetyl trimethyl ammonium bromide (CTAB), or the anionic detergent SDS. Finally, the separation of native membrane protein complexes through the application of blue and clear native gel electrophoresis (BN/CN-PAGE) is reviewed, as well as the free-flow electrophoresis (FFE) of membranes.     

NMR of membrane-associated peptides and proteins

In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purification and crystallization, deposition of membrane protein structures in the Protein Data Bank lags far behind existing structures for soluble proteins. This review describes recent advances in solution NMR allowing the study of a select set of peripheral and integral membrane proteins. Surface-binding proteins discussed include amphitropic proteins, antimicrobial and anticancer peptides, the HIV-1 gp41 peptides, human alpha-synuclein and apolipoproteins. Also discussed are transmembrane proteins including bacterial outer membrane beta-barrel proteins and oligomeric alpha-helical proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. In addition to protein dynamics, protein-lipid interactions such as those between arginines and phosphatidylglycerols have been detected directly by NMR. These examples illustrate the unique role solution NMR spectroscopy plays in structural biology of membrane proteins.     

The contact interface of a 120 kD CheA-CheW complex by methyl TROSY interaction spectroscopy

During bacterial chemotaxis, the histidine autokinase CheA interacts with the chemotaxis receptors with the help of the coupling protein CheW. This interaction is typical of many macromolecular complexes where protein-protein interactions play an important role. In this case, a relatively small protein, CheW, becomes part of a much larger complex. Here we describe a new method to map the residues at a protein-protein interface for macromolecular complexes of molecular weight greater than 100 kD.     

Native analysis of plasma-derived clotting factor VIII concentrates: "sponge effect" and contaminants

In the present study, we analysed two commercially available plasma-derived FVIII preparations, Beriate and Emoclot, through native gel-based approaches (CN-PAGE). The rationale behind this study was to assess whether protein complexes from plasma resisted the aggressive manufacturing processes. A preliminary analysis of plasma complexes was performed focusing on the molecular weight range between 45 kDa and 1 MDa. As a result, we could evidence in both preparations the presence of complexes, which resisted the cryoprecipitation, chromatographic and heat treatment processing steps. These complexes behaved in a "sponge-effect"-like fashion through the enrichment of otherwise less abundant contaminants. The use of an alternative electrophoretic approach such as CN-PAGE allowed us to confirm 2DE-based observations and expand the list of non-FVIII proteins which contaminate the preparations.     

氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展

蛋白质是生命功能的执行者,其功能的发挥受自身结构动态变化、与其他生物分子的相互作用及修饰等因素的调节。因此,对蛋白质及蛋白复合物结构的研究有助于揭示重要生命过程中的分子机理与机制。氢氘交换质谱(Hydrogen deuterium exchange mass spectrometry,HDX-MS)是研究蛋白质结构、动态变化和相互作用的强有力工具,也是传统生物物理手段的重要补充。该文综述了HDX-MS的基本原理、机制、实验方法和研究最新进展,并从蛋白质自身动态变化、蛋白质-小分子相互作用、蛋白质-蛋白质相互作用3个方面介绍了近年来HDX-MS在蛋白及蛋白复合物研究中的应用进展。     

An optimized procedure for solubilization, reduction, and transfer of human breast cancer membrane-enriched fraction by 2-DE

The separation of integral and peripheral membrane proteins is still a challenge, although many achievements have been made in the 2-DE-based membrane proteomics. Using a human breast cancer cell line, MCF-7, we investigated the influences of Tris, reducing reagents, cup loading, and SDS on membrane protein solubilization and separation by 2-DE. The addition of Tris to the sample solution improved the solubilization of the membrane-enriched fraction, and the best-quality gel patterns were obtained at 20 mM Tris. Tributylphosphine (TBP), a reducing agent, was not optimum in the 2-DE process because it not only decreased the solubilization of hydrophobic proteins but also caused some proteins, such as hsp60, prohibitin, and actin, to be resolved to a string of spots. However, when combined with DTT, TBP could improve the resolution of 2-DE patterns. Cup loading significantly facilitated the entrance of membrane proteins into IPG strips and over 1000 protein spots with high resolution were visualized. Adopting this strategy, an ATP synthase alpha chain was resolved into two adjacent spots for the first time in 2-DE gel patterns through the adding DTT in the middle of the IEF. A high SDS concentration in the equilibration buffer enhanced the transfer and increased the staining intensity of 50% of the protein spots in the gels, but also resulted in losses of some spots.     

Monitoring the dynamics of ligand-receptor complexes on model membranes

Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.     

Water-soluble dendritic core-shell-type architectures based on polyglycerol for solubilization of hydrophobic drugs

Since many potential drugs are poorly water soluble, there is a high demand for solubilization agents. Here, we describe the synthesis of dendritic core-shell-type architectures based on hyperbranched polyglycerol for the solubilization of hydrophobic drugs. Amphiphilic macromolecules containing hydrophobic biphenyl groups in the core were synthesized in an efficient three- or four-step procedure by employing Suzuki-coupling reactions. These species were then used to solubilize the commercial drug nimodipine, a calcium antagonist used for the treatment of heart diseases and neurological deficits. Pyrene was also used as a hydrophobic model compound. It turned out that the transport properties of the dendritic polyglycerol derivatives, which are based on hydrophobic host-guest interactions, depend strongly on the degree and type of core functionalization. In the case of the multifunctional nimodipine, additional specific polymer-drug interactions could be tailored by this flexible core design, as detected by UV spectroscopy. The enhancement of solubilization increased 300-fold for nimodipine and 6000-fold for pyrene at a polymer concentration of 10 wt%. The sizes of the polymer-drug complexes were determined by both dynamic light scattering (DLS) experiments and transmission electron microscopy (TEM), and extremely well-defined aggregates with diameters of approximately 10 nm in the presence of a drug were observed. These findings together with a low critical aggregate concentration of 4x10(-6) mol L-1 indicate the controlled self-assembly of the presented amphiphilic dendritic core-shell-type architectures rather than a unimolecular transport behavior.     

Visualization of interactions between siderophore transporters and the energizing protein TonB by native PAGE

Horizontal nondenaturing electrophoresis of proteins in polyacrylamide gels was used to observe specific interactions between membrane proteins. The method was particularly well suited for solubilized transporters of the outer membrane of Gram-negative bacteria, and allowed specific complexes of transporter and the inner-membrane protein TonB to be isolated. We have used this method to investigate the interactions between four different outer-membrane transporters, and the TonB proteins from two different organisms. The results show that a stable complex can be isolated on gels for all the proteins studied, but can depend in some cases of the detergent used for solubilization. Furthermore, we observe cross-species interaction as TonB from a given organism can interact with transporters from another organism.     

Using oxidative crosslinking and proximity labeling to quantitatively characterize protein-protein and protein-Peptide complexes

The quantitative analysis of protein-protein and protein-peptide complexes is of fundamental importance in biochemistry. We report here that nickel-catalyzed proximity biotinylation and Ru(II)(bpy)(3)(2+)-mediated oxidative crosslinking can be used to measure the equilibrium dissociation constant and stoichiometry of protein complexes. Only small amounts of protein are required, neither of the binding partners must be immobilized on a surface, and no special instrumentation is necessary. This chemistry should provide a useful complement to existing methods for the analysis of protein-protein and protein-peptide interactions.     

Flow field-flow fractionation: recent trends in protein analysis

Flow field-flow fractionation (F4) is the gentlest flow-assisted separation technique for analysis of macromolecules. The use of an empty channel as separation device and of a second mobile phase flow as perpendicular field enable F4 to separate analytes under native conditions without any modification of their original structure. Because of this unique peculiarity, F4 has been shown to be ideal for "gentle" separation of biological samples, for example intact proteins and protein complexes, since its early development. Today's F4 is an appealing technique which complements most established separation techniques, for example liquid chromatography and electrophoresis. The number of applications that show the unique advantages of F4 for analysis of protein samples is constantly increasing. In particular, F4 is finding increasing application on very high-molecular-weight species such as protein oligomers, aggregates, and complexes. This review critically discusses recent literature on the application of F4 to proteins. Either stand-alone or coupled with other characterization techniques, F4 is particularly promising for quality control of protein therapeutics, characterization of amyloid proteins, lipoprotein profiling, and as a pre-MS separation step in proteomics.     

Impact of Stable Protein-protein Interaction on Protein Conformational Space

Backbone dihedral angle based clustering approach was applied to investigate the effect of protein complexation on backbone conformational space and the effect on protein dynamics. Three representative enzyme-inhibitor complexes and their comprised proteins were used as models for small-and moderate-sized globular proteins to compare available backbone conformational space before and after complexation. Microsecond time scale molecular dynamic simulations were generated to ensure sufficient statistics. The result suggests that stable protein-protein interactions lead to redistribution of protein backbone mobility and restriction of the protein backbone conformational space, especially for short time scale motions. Surprisingly, these effects are found to be uncorrelated with protein-protein interaction surface. Consistent with many experimental and computational observations, our results indicate that both induced-fit and conformational selection models play roles in stable protein complexation process, with the dominant role being different for different protein complexes.     

Effect of strong detergents and chaotropes on the detection of proteins in two-dimensional gels.

The solubilization of a particular protein is mandatory for its subsequent resolution and detection in two-dimensional gels. However, the extraction solutions, that are compatible with the first-dimensional separation step, such as urea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), do not solubilize all proteins in a sample. We studied the effect of various common, strong detergents and chaotropes, widely used as solubilizing agents, such as sodium dodecyl sulfate, lithium dodecyl sulfate and guanidine hydrochloride, on the solubilization of the total and membrane proteins of the bacterium Haemophilus influenzae. The proteins solubilized with each system were analyzed by two-dimensional electrophoresis and these of interest were identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Use of sodium dodecyl sulfate, lithium dodecyl sulfate or guanidine hydrochloride for the solubilization of total proteins of the microorganism resulted in the detection of several additional spots, representing mainly outer membrane proteins, in comparison with those detected in the soluble protein fraction. Solubilization of the proteins of the cell envelope fraction with sodium dodecyl sulfate did not result in a more efficient protein detection when compared to the extraction with the urea/CHAPS system. When the dry immobilized pH gradient strips were rehydrated in a solution containing the proteins of the membrane fraction solubilized with sodium dodecyl sulfate or lithium dodecyl sulfate, a larger number of protein spots were detected in comparison with strips that were rehydrated in the urea/CHAPS solution. However, no improvement was observed in comparison with protein application in sample cups. The additional proteins detected with the use of strong detergents and chaotropes are in the majority difficult to solubilize and less hydrophobic proteins.     

Delivery of bioactive,gel-isolated proteins into live cells

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.     

Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions

For decades, formaldehyde has been routinely used to cross-link proteins in cells, tissue, and in some instances, even entire organisms. Due to its small size, formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins, DNA, and other reactive molecules. Indeed, formaldehyde cross-linking is an instrumental component of many mainstream analytical/cell biology techniques including chromatin immunoprecipitation (ChIP) of protein-DNA complexes found in nuclei; immunohistological analysis of protein expression and localization within cells, tissues, and organs; and mass spectrometry (MS)-compatible silver-staining methodologies used to visualize low abundance proteins in polyacrylamide gels. However, despite its exquisite suitability for use in the analysis of protein environments within cells, formaldehyde has yet to be commonly employed in the directed analysis of protein-protein interactions and cellular networks. The general purpose of this article is to discuss recent advancements in the use of formaldehyde cross-linking in combination with MS-based methodologies. Key advantages and limitations to the use of formaldehyde over other cross-linkers and technologies currently used to study protein-protein interactions are highlighted, and formaldehyde-based experimental approaches that are proving very promising in their ability to accurately and efficiently identify novel protein-protein and multiprotein interaction complexes are presented.