Transmembrane Prediction Research Articles

Incorporating Community Annotation Interfaces into the CIPRO2.5 Database with Comprehensible Sketches to Support Quick Annotations of Proteome Data.

AbstractUser annotation or comment interfaces are now widely used in several web sites such as journals, news, weblogs and Wikipedia. However, there are only a few biological databases with annotation interfaces. The _Ciona_ intestinalis protein database (CIPRO) was created in order to provide integrated proteome data especially for experimental biologists. The current database contains 89,673 unique sequences covering all the known and predicted gene models. Typical tasks include which gene models are reliable and which function is plausible. The human-curated annotation is most important for the meaningful database.Here we incorporate three new functions into the CIPRO2.5 database (http://cipro.ibio.jp/2.5/), providing enriched resources for the users. First, a community annotation interface as web forms and a user comment editor with rating its comment were added. Second, the web pages were specifically designed to compact for quickly understood overviews. For example, cytolocalization was automatically provided by a color-depicted cell image based on the intensity instead of the numeric values of raw data. The expression data of EST, microarray and 2D-PAGE were integrated as one chart. In addition to these data, the images of transmembrane prediction, domain and motif search, and the OMIM ortholog on the chromosome map were included in each protein page. The last, even the BLAST and PMF search were added to combined fields in the retrieval system.As a result, a total of 11,134 pages were annotated by our community. Furthermore, 2,186 comments were added to the database. Those annotated data are freely accessible at the CIPRO2.5 web site.

CHIKVPRO – a protein sequence annotation database for chikungunya virus

In the recent past, there has been a resurgence of interest in Chikungunya virus (CHIKV) attributed to massive outbreaks of Chikungunya fever in the South-East Asia Region. This has reflected in substantial increase in submission of CHIKV genome sequences to NCBI (National Center for Biotechnology Information) database. Hereby we submit a database "CHIKVPRO" containing structural and functional annotation of Chikungunya virus proteins (25 strains) submitted in the NCBI repository. The CHIKV genome encodes for 9 proteins:4 non-structural and 5 structural. The CHIKVPRO database aims to provide the virology community with a single accession authoritative resource for CHIKV proteome- with reference to physiochemical and molecular properties, proteolytic cleavage sites, hydrophobicity, transmembrane prediction, and classification into functional families using SVMProt and other Expasy tools. The database is freely available at http://www.chikvpro.info/

Lipase Maturation Factor LMF1, Membrane Topology and Interaction with Lipase Proteins in the Endoplasmic Reticulum

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.

Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt

Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.

In Silico Identification of Putative Proton Binding Sites of a Plasma Membrane H+-ATPase Isoform of Arabidopsis Thaliana, AHA1

The plasma membrane potential and secondary transport systems in all eukaryotes are energized by the activity of P-type ATPase membrane proteins: H + ATPase (the proton pump) in plants and fungi and Na + , K + - ATPase (the sodium-potassium pump) in animals. The overall shape of proton pumps has been revealed by electron microscopy. The crystal structure of AHA2, a plasma membrane H + -ATPase isoform of Arabidopsis thaliana, by X-ray crystallography at 3.6 A was available. The isoform is expressed mainly in root. In the present study homology modeling along with transmembrane topology predictions has been used to build the atomic model of AHA1, another plasma membrane H + -ATPase isoform of Arabidopsis thaliana expressing in both root and shoot. AHA2 was used as the template. The homology modeling was done using the MODELLER9v2 software. The model energy was minimized by applying molecular mechanics method. The root mean square deviation (RMSD) for C � atoms between the X-ray and the homology-modeled structures was 0.4 A. Ten transmembrane helices (TM1TM10) in the model were identified. The final model obtained by molecular mechanics and dynamics method was assessed by PROCHECK and VERIFY 3D graph, which showed that the final refined model is reliable. Asp 684 of AHA1 is suggested to act as an essential proton acceptor during proton translocation. The residue may also participate in defining the E 1 proton-binding site. Despite difference in the tissue-specific expression of the two proteins, no remarkable difference in their structure was observed, suggesting that the enzyme isoform evolution may not be linked to its tissue-specific expression.

7TMRmine: a Web server for hierarchical mining of 7TMR proteins

BackgroundSeven-transmembrane region-containing receptors (7TMRs) play central roles in eukaryotic signal transduction. Due to their biomedical importance, thorough mining of 7TMRs from diverse genomes has been an active target of bioinformatics and pharmacogenomics research. The need for new and accurate 7TMR/GPCR prediction tools is paramount with the accelerated rate of acquisition of diverse sequence information. Currently available and often used protein classification methods (e.g., profile hidden Markov Models) are highly accurate for identifying their membership information among already known 7TMR subfamilies. However, these alignment-based methods are less effective for identifying remote similarities, e.g., identifying proteins from highly divergent or possibly new 7TMR families. In this regard, more sensitive (e.g., alignment-free) methods are needed to complement the existing protein classification methods. A better strategy would be to combine different classifiers, from more specific to more sensitive methods, to identify a broader spectrum of 7TMR protein candidates.DescriptionWe developed a Web server, 7TMRmine, by integrating alignment-free and alignment-based classifiers specifically trained to identify candidate 7TMR proteins as well as transmembrane (TM) prediction methods. This new tool enables researchers to easily assess the distribution of GPCR functionality in diverse genomes or individual newly-discovered proteins. 7TMRmine is easily customized and facilitates exploratory analysis of diverse genomes. Users can integrate various alignment-based, alignment-free, and TM-prediction methods in any combination and in any hierarchical order. Sixteen classifiers (including two TM-prediction methods) are available on the 7TMRmine Web server. Not only can the 7TMRmine tool be used for 7TMR mining, but also for general TM-protein analysis. Users can submit protein sequences for analysis, or explore pre-analyzed results for multiple genomes. The server currently includes prediction results and the summary statistics for 68 genomes.Conclusion7TMRmine facilitates the discovery of 7TMR proteins. By combining prediction results from different classifiers in a multi-level filtering process, prioritized sets of 7TMR candidates can be obtained for further investigation. 7TMRmine can be also used as a general TM-protein classifier. Comparisons of TM and 7TMR protein distributions among 68 genomes revealed interesting differences in evolution of these protein families among major eukaryotic phyla.

A unified hydrophobicity scale for multispan membrane proteins

The concept of hydrophobicity is critical to our understanding of the principles of membrane protein (MP) folding, structure, and function. In the last decades, several groups have derived hydrophobicity scales using both experimental and statistical methods that are optimized to mimic certain natural phenomena as closely as possible. The present work adds to this toolset the first knowledge-based scale that unifies the characteristics of both alpha-helical and beta-barrel multispan MPs. This unified hydrophobicity scale (UHS) distinguishes between amino acid preference for solution, transition, and trans-membrane states. The scale represents average hydrophobicity values of amino acids in folded proteins, irrespective of their secondary structure type. We furthermore present the first knowledge-based hydrophobicity scale for mammalian alpha-helical MPs (mammalian hydrophobicity scale--MHS). Both scales are particularly useful for computational protein structure elucidation, for example as input for machine learning techniques, such as secondary structure or trans-membrane span prediction, or as reference energies for protein structure prediction or protein design. The knowledge-based UHS shows a striking similarity to a recent experimental hydrophobicity scale introduced by Hessa and coworkers (Hessa T et al., Nature 2007;450:U1026-U1032). Convergence of two very different approaches onto similar hydrophobicity values consolidates the major differences between experimental and knowledge-based scales observed in earlier studies. Moreover, the UHS scale represents an accurate absolute free energy measure for folded, multispan MPs--a feature that is absent from many existing scales. The utility of the UHS was demonstrated by analyzing a series of diverse MPs. It is further shown that the UHS outperforms nine established hydrophobicity scales in predicting trans-membrane spans along the protein sequence. The accuracy of the present hydrophobicity scale profits from the doubling of the number of integral MPs in the PDB over the past four years. The UHS paves the way for an increased accuracy in the prediction of trans-membrane spans.

AtCSLD2 is an integral Golgi membrane protein with its N-terminus facing the cytosol

Cellulose synthase-like proteins in the D family share high levels of sequence identity with the cellulose synthase proteins and also contain the processive beta-glycosyltransferase motifs conserved among all members of the cellulose synthase superfamily. Consequently, it has been hypothesized that members of the D family function as either cellulose synthases or glycan synthases involved in the formation of matrix polysaccharides. As a prelude to understanding the function of proteins in the D family, we sought to determine where they are located in the cell. A polyclonal antibody against a peptide located at the N-terminus of the Arabidopsis D2 cellulose synthase-like protein was generated and purified. After resolving Golgi vesicles from plasma membranes using endomembrane purification techniques including two-phase partitioning and sucrose density gradient centrifugation, we used antibodies against known proteins and marker enzyme assays to characterize the various membrane preparations. The Arabidopsis cellulose synthase-like D2 protein was found mostly in a fraction that was enriched with Golgi membranes. In addition, versions of the Arabidopsis cellulose synthase-like D2 proteins tagged with a green fluorescent protein was observed to co-localize with a DsRed-tagged Golgi marker protein, the rat alpha-2,6-sialyltransferase. Therefore, we postulate that the majority of Arabidopsis cellulose synthase-like D proteins, under our experimental conditions, are likely located at the Golgi membranes. Furthermore, protease digestion of Golgi-rich vesicles revealed almost complete loss of reaction with the antibodies, even without detergent treatment of the Golgi vesicles. Therefore, the N-terminus of the Arabidopsis cellulose synthase-like D2 protein likely faces the cytosol. Combining this observation with the transmembrane domain predictions, we postulate that the large hydrophilic domain of this protein also faces the cytosol.

In-Depth Investigation of the Soybean Seed-Filling Proteome and Comparison with a Parallel Study of Rapeseed

To better understand the metabolic processes of seed filling in soybean (Glycine max), two complementary proteomic approaches, two-dimensional gel electrophoresis (2-DGE) and semicontinuous multidimensional protein identification technology (Sec-MudPIT) coupled with liquid chromatography-mass spectrometry, were employed to analyze whole seed proteins at five developmental stages. 2-DGE and Sec-MudPIT analyses collectively identified 478 nonredundant proteins with only 70 proteins common to both datasets. 2-DGE data revealed that 38% of identified proteins were represented by multiple 2-DGE species. Identified proteins belonged to 13 (2-DGE) and 15 (Sec-MudPIT) functional classes. Proteins involved in metabolism, protein destination and storage, and energy were highly represented, collectively accounting for 61.1% (2-DGE) and 42.2% (Sec-MudPIT) of total identified proteins. Membrane proteins, based upon transmembrane predictions, were 3-fold more prominent in Sec-MudPIT than 2-DGE. Data were integrated into an existing soybean proteome database (www.oilseedproteomics.missouri.edu). The integrated quantitative soybean database was compared to a parallel study of rapeseed (Brassica napus) to further understand the regulation of intermediary metabolism in protein-rich versus oil-rich seeds. Comparative analyses revealed (1) up to 3-fold higher expression of fatty acid biosynthetic proteins during seed filling in rapeseed compared to soybean; and (2) approximately a 48% higher number of protein species and a net 80% higher protein abundance for carbon assimilatory and glycolytic pathways leading to fatty acid synthesis in rapeseed versus soybean. Increased expression of glycolytic and fatty acid biosynthetic proteins in rapeseed compared to soybean suggests that a possible mechanistic basis for higher oil in rapeseed involves the concerted commitment of hexoses to glycolysis and eventual de novo fatty acid synthesis pathways.

A Generalized Hidden Markov Model Approach to Transmembrane Region Prediction with Poisson Distribution as State Duration Probabilities

We present a novel algorithm to predict transmembrane regions from a primary amino acid sequence. Previous studies have shown that the Hidden Markov Model (HMM) is one of the powerful tools known to predict transmembrane regions; however, one of the conceptual drawbacks of the standard HMM is the fact that the state duration, i.e., the duration for which the hidden dynamics remains in a particular state follows the geometric distribution. Real data, however, does not always indicate such a geometric distribution. The proposed algorithm utilizes a Generalized Hidden Markov Model (GHMM), an extension of the HMM, to cope with this problem. In the GHMM, the state duration probability can be any discrete distribution, including a geometric distribution. The proposed algorithm employs a state duration probability based on a Poisson distribution. We consider the two-dimensional vector trajectory consisting of hydropathy index and charge associated with amino acids, instead of the 20 letter symbol sequences. Also a Monte Carlo method (Forward/Backward Sampling method) is adopted for the transmembrane region prediction step. Prediction accuracies using publicly available data sets show that the proposed algorithm yields reasonably good results when compared against some existing algorithms.

Criteria for confirming sequence periodicity identified by Fourier transform analysis: Application to GCR2, a candidate plant GPCR?

Methods to determine peridiodicity in protein sequences are useful for inferring function. Fourier transformation is one approach but care is required to ensure the periodicity is genuine. Here we have shown that empirically-derived statistical tables can be used as a measure of significance. Genuine protein sequences data rather than randomly generated sequences were used as the statistical backdrop. The method has been applied to G-protein coupled receptor (GPCR) sequences, by Fourier transformation of hydrophobicity values, codon frequencies and the extent of over-representation of codon pairs; the latter being related to translational step times. Genuine periodicity was observed in the hydrophobicity whereas the apparent periodicity (as inferred from previously reported measures) in the translation step times was not validated statistically. GCR2 has recently been proposed as the plant GPCR receptor for the hormone abscisic acid. It has homology to the Lanthionine synthetase C-like family of proteins, an observation confirmed by fold recognition. Application of the Fourier transform algorithm to the GCR2 family revealed strongly predicted seven fold periodicity in hydrophobicity, suggesting why GCR2 has been reported to be a GPCR, despite negative indications in most transmembrane prediction algorithms. The underlying multiple sequence alignment, also required for the Fourier transform analysis of periodicity, indicated that the hydrophobic regions around the 7 GXXG motifs commence near the C-terminal end of each of the 7 inner helices of the α-toroid and continue to the N-terminal region of the helix. The results clearly explain why GCR2 has been understandably but erroneously predicted to be a GPCR.

Genetic characterization reveals no role for the reported ABA receptor, GCR2, in ABA control of seed germination and early seedling development in Arabidopsis

Abscisic acid (ABA) is perceived by several different types of receptors in plant cells. At the cell surface, the ABA signal is proposed to be perceived by GCR2, which mediates ABA responses in seed germination, early seedling development and stomatal movement. GCR2 was also proposed to be a seven-transmembrane (7TM) G-protein-coupled receptor (GPCR). Here we characterize GCR2 and one of its two homologs, GCR2-LIKE 1 (GCL1), in ABA-mediated seed germination and early seedling development in Arabidopsis. We show that loss-of-function mutations in GCL1 did not confer ABA insensitivity. Similarly, we did not observe ABA insensitivity in three independent gcr2 alleles. Furthermore, we generated gcr2 gcl1 double mutants and found that the double mutants still had near wild-type responses to ABA. Consistent with this, we found that the transcription of ABA marker genes was induced by ABA to levels that were comparable in wild type and gcr2 and gcl1 single and double mutants. On the other hand, the loss-of-function alleles of the sole Arabidopsis heterotrimeric G protein alpha subunit, GPA1, were hypersensitive to ABA in the ABA-inhibition of seed germination and early seedling development, disfavoring a genetic coupling of GCR2 by GPA1. Using multiple robust transmembrane prediction systems, GCR2 was predicted not to be a 7TM protein, a structural hallmark of GPCRs. Taken together, our results do not support the notion that GCR2 is an ABA-signaling GPCR in seed germination and early seedling development.

Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server

When using conventional transmembrane topology and signal peptide predictors, such as TMHMM and SignalP, there is a substantial overlap between these two types of predictions. Applying these methods to five complete proteomes, we found that 30–65% of all predicted signal peptides and 25–35% of all predicted transmembrane topologies overlap. This impairs predictions of 5–10% of the proteome, hence this is an important issue in protein annotation.To address this problem, we previously designed a hidden Markov model, Phobius, that combines transmembrane topology and signal peptide predictions. The method makes an optimal choice between transmembrane segments and signal peptides, and also allows constrained and homology-enriched predictions.We here present a web interface (http://phobius.cgb.ki.se and http://phobius.binf.ku.dk) to access Phobius.

Characterization of glycosomal RING finger proteins of trypanosomatids

The glycosomes of trypanosomatids are essential organelles that are evolutionarily related to peroxisomes of other eukaryotes. The peroxisomal RING proteins—PEX2, PEX10 and PEX12—comprise a network of integral membrane proteins that function in the matrix protein import cycle. Here, we describe PEX10 and PEX12 in Trypanosoma brucei, Leishmania major, and Trypanosoma cruzi. We expressed GFP fusions of each T. brucei coding region in procyclic form T. brucei, where they localized to glycosomes and behaved as integral membrane proteins. Despite the weak transmembrane predictions for TbPEX12, protease protection assays demonstrated that both the N and C termini are cytosolic, similar to mammalian PEX12. GFP fusions of T. cruzi PEX10 and L. major PEX12 also localized to glycosomes in T. brucei indicating that glycosomal membrane protein targeting is conserved across trypanosomatids.

Both Cleavage Products of the mCLCA3 Protein Are Secreted Soluble Proteins

Members of the chloride channels, calcium-activated (CLCA) family of proteins and in particular the murine mCLCA3 (alias gob-5) and its human ortholog hCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. Initial studies have indicated that these proteins evoke a calcium-activated chloride conductance when transfected into human embryonic kidney cells 293 cells. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unknown chloride channels. Here, we present a systematic biochemical analysis of the posttranslational processing and intracellular trafficking of the mCLCA3 protein. Pulse-chase experiments after metabolic protein labeling of mCLCA3-transfected COS-1 or human embryonic kidney 293 cells revealed cleavage of a primary 110-kDa mCLCA3 translation product in the endoplasmic reticulum into a 75-kDa amino-terminal and a 35-kDa carboxyl-terminal protein that were glycosylated and remained physically associated with each other. Confocal fluorescent analyses identified both cleavage products in vesicles of the secretory pathway. Neither cleavage product was associated with the cell membrane at any time. Instead, both subunits were fully secreted into the extracellular environment as a soluble complex of two glycoproteins. These results suggest that the two mCLCA3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules. Furthermore, our results point toward significant structural differences between mCLCA3 and its human ortholog, hCLCA1, which is thought to be a single, non-integral membrane protein.

Expanding the Subproteome of the Inner Mitochondria Using Protein Separation Technologies

Currently no single proteomics technology has sufficient analytical power to allow for the detection of an entire proteome of an organelle, cell, or tissue. One approach that can be used to expand proteome coverage is the use of multiple separation technologies especially if there is minimal overlap in the proteins observed by the different methods. Using the inner mitochondrial membrane subproteome as a model proteome, we compared for the first time the ability of three protein separation methods (two-dimensional liquid chromatography using the ProteomeLab PF 2D Protein Fractionation System from Beckman Coulter, one-dimensional reversed phase high performance liquid chromatography, and two-dimensional gel electrophoresis) to determine the relative overlap in protein separation for these technologies. Data from these different methods indicated that a strikingly low number of proteins overlapped with less than 24% of proteins common between any two technologies and only 7% common among all three methods. Utilizing the three technologies allowed the creation of a composite database totaling 348 non-redundant proteins. 82% of these proteins had not been observed previously in proteomics studies of this subproteome, whereas 44% had not been identified in proteomics studies of intact mitochondria. Each protein separation method was found to successfully resolve a unique subset of proteins with the liquid chromatography methods being more suited for the analysis of transmembrane domain proteins and novel protein discovery. We also demonstrated that both the one- and two-dimensional LC allowed for the separation of the alpha-subunit of F1F0 ATP synthase that differed due to a change in pI or hydrophobicity.

The MPI Bioinformatics Toolkit for protein sequence analysis

The MPI Bioinformatics Toolkit is an interactive web service which offers access to a great variety of public and in-house bioinformatics tools. They are grouped into different sections that support sequence searches, multiple alignment, secondary and tertiary structure prediction and classification. Several public tools are offered in customized versions that extend their functionality. For example, PSI-BLAST can be run against regularly updated standard databases, customized user databases or selectable sets of genomes. Another tool, Quick2D, integrates the results of various secondary structure, transmembrane and disorder prediction programs into one view. The Toolkit provides a friendly and intuitive user interface with an online help facility. As a key feature, various tools are interconnected so that the results of one tool can be forwarded to other tools. One could run PSI-BLAST, parse out a multiple alignment of selected hits and send the results to a cluster analysis tool. The Toolkit framework and the tools developed in-house will be packaged and freely available under the GNU Lesser General Public Licence (LGPL). The Toolkit can be accessed at .

Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus.

Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unusually diverse. In addition to flagellin, PibD cleaves three sugar-binding proteins unique to this species and a number of proteins with unknown function. Here we demonstrate that PibD contains two aspartic acid residues that are essential for cleavage activity. An additional pair of aspartic acids in a large cytoplasmic loop is also important for function and is possibly involved in leader peptide recognition. Combining the results of transmembrane segment predictions and cysteine-labeling experiments, we suggest a membrane topology model for PibD with the active-site aspartic acid residues exposed to the cytosol.

Analysis of Membrane Proteins from Human Chronic Myelogenous Leukemia Cells: Comparison of Extraction Methods for Multidimensional LC−MS/MS

An important strategy for "shotgun proteomics" profiling involves solution proteolysis of proteins, followed by peptide separation using multidimensional liquid chromatography and automated sequencing by mass spectrometry (LC-MS/MS). Several protocols for extracting and handling membrane proteins for shotgun proteomics experiments have been reported, but few direct comparisons of different protocols have been reported. We compare four methods for preparing membrane proteins from human cells, using acid labile surfactants (ALS), urea, and mixed organic-aqueous solvents. These methods were compared with respect to their efficiency of protein solubilization and proteolysis, peptide and protein recovery, membrane protein enrichment, and peptide coverage of transmembrane proteins. Overall, approximately 50-60% of proteins recovered were membrane-associated, identified from Gene Ontology annotations and transmembrane prediction software. Samples extracted with ALS, extracted with urea followed by dilution, or extracted with urea followed by desalting yielded comparable peptide recoveries and sequence coverage of transmembrane proteins. In contrast, suboptimal proteolysis was observed with organic solvent. Urea extraction followed by desalting may be a particularly useful approach, as it is less costly than ALS and yields satisfactory protein denaturation and proteolysis under conditions that minimize reactivity with urea-derived cyanate. Spectral counting was used to compare datasets of proteins from membrane samples with those of soluble proteins from K562 cells, and to estimate fold differences in protein abundances. Proteins most highly abundant in the membrane samples showed enrichment of integral membrane protein identifications, consistent with their isolation by differential centrifugation.

Topology and boundaries of the aerotaxis receptor Aer in the membrane of Escherichia coli.

Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4 degrees C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4'-maleimidylstilbene-2, 2' disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.