TransPath: a computational method for locating ion transit pathways through membrane proteins

The finely tuned structures of membrane channel proteins allow selective passage of ions through the available aqueous pores. To understand channel function, it is crucial to locate the pores and study their physical and chemical properties. Here, we propose a new pore-searching algorithm (TransPath), which uses the Configurational Bias Monte Carlo (CBMC) method to generate transmembrane trajectories driven by both geometric and electrostatic features. The trajectories are binned into groups determined by a vector distance criterion. From each group, a representative trajectory is selected based on the Rosenbluth weight, and the geometrically optimal path is obtained by simulated annealing. Candidate ion pathways can then be determined by analysis of the radius and potential profiles. The proposed method and its implementation are illustrated using the bacterial KcsA potassium channel as an example. The procedure is then applied to the more complex structures of the bacterial E. coli chloride channel homolog and a homology model of the ClC-0 channel.     

Muscarinic receptors: A comparative analysis of structural features and binding modes through homology modelling and molecular docking

Three-dimensional models of the five human muscarinic receptors were obtained from their known sequences. Homology modelling based on the crystallographic structure of bovine rhodopsin yielded models compatible with known results from site-directed mutagenesis studies. The only exceptions were the cytoplasmic loop 3 (CL3) in the five receptors, and the large C-terminal domain in M(1). Here, homology modelling with other closely related proteins allowed to solve these gaps. A detailed comparative discussion of the five models is given. The second part of the work involved docking experiments with the physiological ligand acetylcholine, again yielding results entirely compatible with results from mutagenesis experiments. The study revealed analogies and differences between the five receptors in the residues, and interactions leading to the recognition and binding of acetylcholine.     

Proteomic analysis of rat hippocampal plasma membrane: characterization of potential neuronal-specific plasma membrane proteins

The hippocampus is a distinct brain structure that is crucial in memory storage and retrieval. To identify comprehensively proteins of hippocampal plasma membrane (PM) and detect the neuronal-specific PM proteins, we performed a proteomic analysis of rat hippocampus PM using the following three technical strategies. First, proteins of the PM were purified by differential and density-gradient centrifugation from hippocampal tissue and separated by one-dimensional electophoresis, digested with trypsin and analyzed by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) tandem mass spectrometry (MS/MS). Second, the tryptic peptide mixture from PMs purified from hippocampal tissue using the centrifugation method was analyzed by liquid chromatography ion-trap ESI-MS/MS. Finally, the PM proteins from primary hippocampal neurons purified by a biotin-directed affinity technique were separated by one-dimensional electrophoresis, digested with trypsin and analyzed by ESI-Q-TOF-MS/MS. A total of 345, 452 and 336 non-redundant proteins were identified by each technical procedure respectively. There was a total of 867 non-redundant protein entries, of which 64.9% are integral membrane or membrane-associated proteins. One hundred and eighty-one proteins were detected only in the primary neurons and could be regarded as neuronal PM marker candidates. We also found some hypothetical proteins with no functional annotations that were first found in the hippocampal PM. This work will pave the way for further elucidation of the mechanisms of hippocampal function.     

Signaling through sphingolipid microdomains of the plasma membrane: The concept of signaling platform

Transmembrane signaling requires modular interactions between signaling proteins, phosphorylation or dephosphorylation of the interacting protein partners [1] and temporary elaboration of supramolecular structures [2], to convey the molecular information from the cell surface to the nucleus. Such signaling complexes at the plasma membrane are instrumental in translating the extracellular cues into intracellular signals for gene activation. In the most straightforward case, ligand binding promotes homodimerization of the transmembrane receptor which facilitates modular interactions between the receptor's cytoplasmic domains and intracellular signaling and adaptor proteins [3]. For example, most growth factor receptors contain a cytoplasmic protein tyrosine kinase (PTK) domain and ligand-mediated receptor dimerization leads to cross phosphorylation of tyrosines in the receptor's cytoplasmic domains, an event that initiates the signaling cascade [4]. In other signaling pathways where the receptors have no intrinsic kinase activity, intracellular non-receptor PTKs (i.e. Src family PTKs, JAKs) are recruited to the cytoplasmic domain of the engaged receptor. Execution of these initial phosphorylations and their translation into efficient cellular stimulation requires concomitant activation of diverse signaling pathways. Availability of stable, preassembled matrices at the plasma membrane would facilitate scaffolding of a large array of receptors, coreceptors, tyrosine kinases and other signaling and adapter proteins, as it is the case in signaling via the T cell antigen receptor [5]. The concept of the signaling platform [6] has gained usage to characterize the membrane structure where many different membrane-bound components need to be assembled in a coordinated manner to carry out signaling.The structural basis of the signaling platform lies in preferential assembly of certain classes of lipids into distinct physical and functional compartments within the plasma membrane [7,8]. These membrane microdomains or rafts (Figure 1) serve as privileged sites where receptors and proximal signaling molecules optimally interact [9]. In this review, we shall discuss first how signaling platforms are assembled and how receptors and their signaling machinery could be functionally linked in such structures. The second part of our review will deal with selected examples of raft-based signaling pathways in T lymphocytes and NK cells to illustrate the ways in which rafts may facilitate signaling.     

Immunochemical purification of probe-labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.     

ATP conformations and ion binding modes in the active site of anthrax edema factor: A computational analysis

The Edema Factor (EF), one of the virulence factors of anthrax, is an adenylyl cyclase that promotes the overproduction of cyclic‐AMP (cAMP) from ATP, and therefore perturbs cell signaling. Crystallographic structures of EF bound to ATP analogs and reaction products, cyclic‐AMP, and Pyrophosphate (PPi), revealed different substrate conformations and catalytic‐cation binding modes, one or two cations being observed in the active site. To shed light into the biological significance of these crystallographic structures, the energetics, geometry, and dynamics of the active site are analyzed using molecular dynamics simulations. The ATP conformation observed in the one‐metal‐ion structure allows stronger interactions with the catalytic ion, and ATP is more restrained than in the structure containing two Mg²⁺ ions. Therefore, we propose that the conformation observed in the one‐ion crystal structure is a more probable starting point for the reaction. The simulations also suggest that a C3′‐endo sugar pucker facilitates nucleophilic attack. Additionally, the two‐cation binding mode restrains the mobility of the reaction products, and thus their tendency to dissociate. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of surface binding sites in glycoside hydrolases: A computational study

Structural properties of carbohydrate surface binding sites (SBSs) were investigated with computational methods. Eighty‐five SBSs of 44 enzymes in 119 Protein Data Bank (PDB) files were collected as a dataset. On the basis of SBSs shape, they were divided into 3 categories: flat surfaces, clefts, and cavities (types A, B, and C, respectively). Ligand varieties showed the correlation between shape of SBSs and ligands size. To reduce cut‐off differences in each SBSs with different ligand size, molecular docking were performed. Molecular docking results were used to refine SBSs classification and binding sites cut‐off. Docking results predicted putative ligands positions and displayed dependence of the ligands binding mode to the structural type of SBSs. Physicochemical properties of SBSs were calculated for all docking results with YASARA Structure. The results showed that all SBSs are hydrophilic, while their charges could vary and depended to ligand size and defined cut‐off. Surface binding sites type B had highest average values of solvent accessible surface area. Analysis of interactions showed that hydrophobic interactions occur more than hydrogen bonds, which is related to the presence of aromatic residues and carbohydrates interactions.     

Cell wall proteomic of Brachypodium distachyon grains: A focus on cell wall remodeling proteins

Cell walls play key roles during plant development. Following their deposition into the cell wall, polysaccharides are continually remodeled according to the growth stage and stress environment to accommodate cell growth and differentiation. To date, little is known concerning the enzymes involved in cell wall remodeling, especially in gramineous and particularly in the grain during development. Here, we investigated the cell wall proteome of the grain of Brachypodium distachyon. This plant is a suitable model for temperate cereal crops. Among the 601 proteins identified, 299 were predicted to be secreted. These proteins were distributed into eight functional classes; the class of proteins that act on carbohydrates was the most highly represented. Among these proteins, numerous glycoside hydrolases were found. Expansins and peroxidases, which are assumed to be involved in cell wall polysaccharide remodeling, were also identified. Approximately half of the proteins identified in this study were newly discovered in grain and were not identified in the previous proteome analysis conducted using the culms and leaves of B. distachyon. Therefore, the data obtained from all organs of B. distachyon infer a global cell wall proteome consisting of 460 proteins. At present, this is the most extensive cell wall proteome of a monocot species.     

Phosphorylation-dependent C-terminal binding of 14-3-3 proteins promotes cell surface expression of HIV co-receptor GPR15

Membrane trafficking is dictated by dynamic molecular interactions involving discrete determinants in the cargo proteins and the intracellular transport machineries. We have previously reported that cell surface expression of GPR15, a G protein-coupled receptor (GPCR) that serves as a co-receptor for HIV, is correlated with the mode III binding of 14-3-3 proteins to the receptor C terminus. Here we provide a mechanistic basis for the role of 14-3-3 in promoting the cell surface expression of GPR15. The Ala mutation of penultimate phospho-Ser (S359A) that abolishes 14-3-3 binding resulted in substantially reduced O-glycosylation and the cell surface expression of GPR15. The surface membrane protein CD8 fused with the C-terminal tail of GPR15(S359A) mutant was re-localized in the endoplasmic reticulum (ER). In the context of S359A mutation, the additional mutations in the upstream stretch of basic residues (RXR motif) restored O-glycosylation and the cell surface expression. The RXR motif was responsible for the interaction with coatomer protein I (COPI), which was inversely correlated with the 14-3-3 binding and cell surface expression. These results suggest that 14-3-3 binding promotes cell surface expression of GPR15 by releasing the receptor from ER retrieval/retention pathway that is mediated by the interaction of RXR motif and COPI. Moreover, 14-3-3 binding substantially increased the stability of GPR15 protein. Thus 14-3-3 proteins play multiple roles in biogenesis and trafficking of an HIV co-receptor GPR15 to control its cell surface density in response to the phosphorylation signal.     

Proteomic analysis of mouse liver plasma membrane: use of differential extraction to enrich hydrophobic membrane proteins

To comprehensively identify proteins of liver plasma membrane (PM), we isolated PMs from mouse liver by sucrose density gradient centrifugation. An optimized extraction method for whole PM proteins and several methods of differential extraction expected to enrich hydrophobic membrane proteins were tested. The extracted PM proteins were separated by 2-DE, and were identified by MALDI-TOF-MS, and ESI-quadrupole-TOF MS. As the complementary method, 1-DE-MS/MS was also used to identify PM proteins. The optimized lysis buffer containing urea, thiourea, CHAPS and NP-40 was able to extract more PM proteins, and treatment of PM samples with chloroform/methanol and sodium carbonate led to enrichment of more hydrophobic PM proteins. From the mouse liver PM fraction, 175 non-redundant gene products were identified, of which 88 (about 50%) were integral membrane proteins with one to seven transmembrane domains. The remaining products were probably membrane-associated and cytosolic proteins. The function distribution of all the identified liver PM proteins was analyzed; 40% represented enzymes, 12% receptors and 9% proteins with unknown function.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

A laser microsurgical method of cell wall removal allows detection of largeconductance ion channels in the guard cell plasma membrane

Summary Application of patch clamp techniques to higher-plant cells has been subject to the limitation that the requisite contact of the patch electrode with the cell membrane necessitates prior enzymatic removal of the plant cell wall. Because the wall is an integral component of plant cells, and because cell-wall-degrading enzymes can disrupt membrane properties, such enzymatic treatments may alter ion channel behavior. We compared ion channel activity in enzymatically isolated protoplasts ofVicia faba guard cells with that found in membranes exposed by a laser microsurgical technique in which only a tiny portion of the cell wall is removed while the rest of the cell remains intact within its tissue environment. “Laserassisted” patch clamping reveals a new category of high-conductance (130 to 361 pS) ion channels not previously reported in patch clamp studies on plant plasma membranes. These data indicate that ion channels are present in plant membranes that are not detected by conventional patch clamp techniques involving the production of individual plant protoplasts isolated from their tissue environment by enzymatic digestion of the cell wall. Given the large conductances of the channels revealed by laser-assisted patch clamping, we hypothesize that these channels play a significant role in the regulation of ion content and electrical signalling in guard cells.     

Understanding the interaction of proteins to ion exchange chromatographic supports: A surface energetics approach

Ion exchange chromatography is one of the most widely used chromatographic technique for the separation and purification of important biological molecules. Due to its wide applicability in separation processes, a targeted approach is required to suggest the effective binding conditions during ion exchange chromatography. A surface energetics approach was used to study the interaction of proteins to different types of ion exchange chromatographic beads. The basic parameters used in this approach are derived from the contact angle, streaming potential, and zeta potential values. The interaction of few model proteins to different anionic and cationic exchanger, with different backbone chemistry, that is, agarose and methacrylate, was performed. Generally, under binding conditions, it was observed that proteins having negative surface charges showed strong to lose interaction (20 kT for Hannilase to 0.5 kT for IgG) with different anionic exchangers (having different positive surface charges). On the contrary, anionic exchangers showed almost no interaction (0–0.1 kT) with the positively charged proteins. An inverse behavior was observed for the interaction of proteins to cationic exchangers. The outcome from these theoretical calculations can predict the binding behavior of different proteins under real ion exchange chromatographic conditions. This will ultimately propose a better bioprocess design for protein separation.     

A novel method for isolation of membrane proteins: a baculovirus surface display system

We have developed a novel baculovirus surface display (BVSD) system for the isolation of membrane proteins. We expressed a reporter gene that encoded hemagglutinin gene fused in frame with the signal peptide and transmembrane domain of the baculovirus gp64 protein, which is displayed on the surface of BmNPV virions. The expression of this fusion protein on the virion envelope allowed us to develop two methods for isolating membrane proteins. In the first method, we isolated proteins directly from the envelope of budding BmNPV virions. In the second method, we isolated proteins from cellular membranes that had disintegrated due to viral egress. We isolated 6756 proteins. Of these, 1883 have sequence similarities to membrane proteins and 1550 proteins are homologous to known membrane proteins. This study indicates that membrane proteins can be effectively isolated using our BVSD system. Using an analogous method, membrane proteins can be isolated from other eukaryotic organisms, including human beings, by employing a host cell-specific budding virus.     

Electrophoretic analysis of the plasma membrane proteins of a mutant of Neurospora crassa which lacks a cell wall

The plasma membrane proteins of a mutant of Neurospora crassa (FGSC No. 326) which lacks a cell wall were analyzed by two-dimensional polyacrylamide gel electrophoresis. Approximately 180 different proteins were detected in purified plasma membrane preparations. Nonpermeant labeling experiments indicated that approximately 40% of these proteins were exposed on the extracytoplasmic surface of the plasma membranes of these cells. The studies demostrate the complexity of the protein composition of N. crassa 326 plasma membranes to be greater than has been suggested by previous investigations.     

Differential arrival of newly synthesized apical and basolateral plasma membrane proteins in the epithelial cell line A6

The labeling of specific cell surface proteins with biotin was used to examine both protein distribution and delivery of newly synthesized proteins to the apical and basolateral cell surface in A6 cells. Steady-state metabolic labeling with [³⁵S]methionine followed by specific cell surface biotinylation demonstrated polarization of membrane proteins. The delivery of newly synthesized proteins to the apical or basolateral cell surface was examined by metabolic labeling with [³⁵S]methionine using a pulse-chase protocol in combination with specific cell surface biotinylation. Newly synthesized biotinylated proteins at the apical cell surface reached a maximum after a 5 min chase, and then fell over the remainder of a 2 hr chase. The bulk flow of newly synthesized proteins to the basolateral membrane slowly rose to a maximum after 90 min. The detergent Triton X-114 was used to examine delivery of hydrophilic and hydrophobic proteins to the cell surface. Delivery of both hydrophilic and hydrophobic proteins to the apical cell surface reached a maximum 5 to 10 min into the chase period. The arrival of hydrophilic proteins at the basolateral surface showed early delivery and a maximum peak delivery at 120 min into the chase period. In contrast, only an early peak of delivery of newly synthesized hydrophobic proteins to the basolateral membrane was observed.This work was supported by grants from the American Heart Association, the National Kidney Foundation of the Delaware Valley, and from the Department of Veterans Affairs. T.R.K. is a recipient of an Established Investigatorship Award from the American Heart Association.     

Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins: Antagonistic eeffects of cyclic AMP and cyclic GMP

³²P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E₁ one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP.Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes.These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.     

Characterizing the noncovalent binding behavior of tartrazine to lysozyme: A combined spectroscopic and computational analysis

Tartrazine is a stable water‐soluble azo dye widely used as a food additive, which could pose potential threats to humans and the environment. In this paper, we evaluated the response mechanism between tartrazine and lysozyme under simulated conditions by means of biophysical methods, including multiple spectroscopic techniques, isothermal titration calorimetry (ITC), and molecular docking studies. From the multispectroscopic analysis, we found that tartrazine could effectively quench the intrinsic fluorescence of lysozyme to form a complex and lead to the conformational and microenvironmental changes of the enzyme. The ITC measurements suggested that the electrostatic forces played a major role in the binding of tartrazine to lysozyme with two binding sites. Finally, the molecular docking indicated that tartrazine had specific interactions with the residues of Trp108. The study provides an important insight within the binding mechanism of tartrazine to lysozyme in vitro.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.