Elucidating in Vivo Structural Dynamics in Integral Membrane Protein by Hydroxyl Radical Footprinting

We describe here a novel footprinting technique to probe the in vivo structural dynamics of membrane protein. This method utilized in situ generation of hydroxyl radicals to oxidize and covalently modify biomolecules on living Escherichia coli cell surface. After enriching and purifying the membrane proteome, the modified amino acid residues of the protein were identified with tandem mass spectrometry to map the solvent-accessible surface of the protein that will form the footprint of in vivo structure of the protein. Of about 100 outer membrane proteins identified, we investigated the structure details of a typical β-barrel structure, the porin OmpF. We found that six modified tryptic peptides of OmpF were reproducibly detected with 19 amino acids modified under the physiological condition. The modified amino acid residues were widely distributed in the external loop area, β-strands, and periplasmic turning area, and all of them were validated as solvent-accessible according to the crystallography data. We further extended this method to study the dynamics of the voltage gating of OmpF in vivo using mimic changes of physiological circumstance either by pH or by ionic strength. Our data showed the voltage gating of porin OmpF in vivo for the first time and supported the proposed mechanism that the local electrostatic field changes in the eyelet region may alter the porin channels to switch. Thus, this novel method can be a potentially efficient method to study the structural dynamics of the membrane proteins of a living cell.One of the most challenging problems in biological sciences is the correlation of the dynamic three-dimensional (3D)¹ structural changes in membrane proteins to their biological functions. Comprising about 30% of the human proteome, membrane proteins are critical mediators of material and information transfer between cells and their environment and are targets for many growth factors and pharmacologically active compounds. Signals from binding of ligands or drugs to receptors are transduced through conformational changes in the receptors. Therefore, understanding the dynamic conformational changes in the structure of membrane proteins is essential to the understanding of many biological processes and has important implications in human health.Although some methodologies including NMR and x-ray have been developed to study protein structures in atomic resolution, the determination of the structures of integral membrane proteins (IMPs) remains one of the most challenging problems in biological sciences (1). IMPs are usually insoluble low abundance proteins for which expression, purification, and crystallization are generally too inefficient to generate sufficient materials for conventional structural analysis techniques such as NMR and x-ray. Additionally, IMPs in living cells are constantly interacting with different molecules depending on the external environment and biological states of the cell such that the conformation of IMPs is highly dynamic. Hence monitoring real time conformational changes in these proteins by NMR and x-ray methods is unfeasible, and alternative methods are needed.Recent progress in MS has enabled a novel MS-based protein oxidative footprinting technique to determine structural information by mapping of oxidation induced by hydroxyl (OH) radicals. This method is an adaptation of the OH radical footprinting first developed by Tullius and co-workers (2, 3) for the folding study of DNA/RNA molecules in solution. Several groups have since extended this method in combination with mass spectrometry for the mapping of a protein surface (4–8). In these studies, OH radicals oxidize amino acid residues located on the protein surface and produce stable covalent modifications to side chains without causing backbone cleavages. Due to the very small size and nonspecific activity of hydroxyl radicals, this is a random process dependent only on the solvent-accessible surface and the chemical properties of the exposed amino acids. It has been reported that there are about 12 possible types of side-chain oxidation products in protein footprinting experiments (9); however, not all of these oxidation products are common events as reviewed. The most common event results in formation of an alcohol group for almost all the amino acid residues with mass increases of +16 Da. Another common event is the formation of an aldehyde/ketone group for eight residues, Val, Ile, Leu, Lys, Arg, Pro, Glu, and Gln, with mass increases of +14 Da (9). Others include +32 Da on Cys, Met, Trp, Phe, and Tyr; +48 Da on Cys and Trp; +5, −10, −22, and −23 Da on His; −30 Da on Asp and Glu; −16 Da on Cys; −32 Da on Met; and −2 Da on Ser and Thr (9).The oxidized protein is subsequently sequenced by MS/MS to locate the oxidized amino acid residues. The oxidized amino acid residues provide the surface information of the protein, and thus the surface topology can be mapped. At the same time, the oxidation level of each side chain can be accurately measured by quantitative liquid chromatography-coupled MS. Because the level of oxidation for each tryptic peptide depends primarily on the solvent accessibility of the peptide side chains that is in turn dependent on the conformation of the protein, the oxidation level and changes in its level for each peptide can therefore be used to determine conformation and conformational changes of the protein. This approach has been shown to be a powerful technique in understanding ligand-induced conformational changes when coupled with existing structural data that are either experimentally or computationally generated (10).Despite recent advances, most current surface mapping techniques still require purified proteins and are therefore not amenable to the conformation determination of most IMPs or their complexes. In particular, the current techniques still cannot determine real time in vivo structural changes of IMPs that are important in understanding the functions of IMPs. Therefore, the structural dynamics of IMP remain elusive.IMPs display two membrane-spanning tertiary structural motifs, i.e. α-helix bundles in the cytoplasmic membrane and β-barrels in the outer membrane (11). In Gram-negative bacteria, the outer membrane forms a protective permeability barrier around the cells and serves as a molecular filter for hydrophilic substances (12). To facilitate this, channel-forming proteins are embedded in the outer membrane to mediate the transport of nutrients and ions across the membrane into the periplasm. The most abundant proteins in this class are the porins that form aqueous passive channels with a physical diameter of about 1 nm for the transport of ions and relatively small polar molecules across the outer membrane of bacteria (13–15). Porins form β-barrels with 16 or 18 membrane-spanning antiparallel β-strands that are connected by short turns on the periplasmic side named T1, T2, etc. and long loops on the extracellular side named L1, L2, etc. (16, 17). In all porins, the constriction at the barrel center is formed by an inserted loop L3 (13, 16, 18), which is not exposed to the cell surface but folds back into the channel and contributes significantly to the permeability of the pore. Functional porins are homotrimers of these β-barrel subunits with each subunit producing a channel in the outer membrane, and the trimer therefore contains three channels (14). Currently known porins fall into two distinct groups: 16-stranded general diffusion porins such as the matrix porin OmpF that transports ions and small molecules (<600 Da) without much selectivity and 18-stranded specific porins such as maltoporin that have a precise selectivity for a defined substrate (15, 17, 19–22). General porin trimers exhibit symmetrical or asymmetrical voltage gating when reconstituted into planar lipid bilayers in electrophysiological studies (23–27). By measuring the ion conductance in vitro, porin trimers were found to be able to exhibit at least two functional states, an open and a closed state, in response to changes in the transmembrane potential difference, known as “voltage gating”, and the voltage above which the porin channel will be closed is called the critical voltage, Vc. Although existing data generally support voltage gating in vitro, there are no data to support voltage gating in vivo (25, 27).We describe here a novel adaptation of an oxidative footprinting and MS technique to study in vivo conformational changes of IMPs in living cells. We used Escherichia coli as a model to study the structural dynamics of the outer membrane proteins with an emphasis on the matrix porin OmpF. Considering that the yellowish and cloudy E. coli bacteria culture would interfere with the laser photolysis of H₂O₂ because H₂O₂ only absorbs laser light at a wavelength around 250 nm and this cloudy medium would inhibit the laser penetration (4), we adopted a Fenton reaction but not the pulsed laser photolysis method to oxidize the living bacteria. Fenton oxidation is not guaranteed for many proteins, especially the highly dynamic proteins, because this method needs a relatively longer incubation time (2, 4, 5, 9). This time scale is too long because most of the conformational changes of macromolecules take place only in the time scale of milliseconds to seconds (10). However, Fenton chemistry is suitable to study the outer membrane porins because they have extremely stable structures that are restricted by both the intramolecular hydrogen bond and the hydrophobic interaction with the lipid bilayer wall, and a large conformational change of this kind of protein is considered unlikely (14, 20).In this method, living E. coli cells were first exposed to OH radicals generated from an in situ Fenton reaction between hydrogen peroxide and Fe(II)-bound EDTA. The outer membrane proteins that have a large solvent-accessible surface area would be preferentially oxidized by the OH radicals. The outer membrane proteins were then isolated and enriched from the cell lysate. The porin OmpF was subsequently purified by 1D SDS-PAGE electrophoresis followed by proteolysis to generate peptides for analysis and identification by LC-MS/MS and bioinformatics analysis. The structural information of porin OmpF as interpreted from our oxidative footprinting and MS study was in agreement with the x-ray crystallography structure (20). Highly reproducible oxidized amino acid residues were widely distributed in the external loops, transmembrane β-strands, and periplasmic turnings of the protein, and all of them were validated as solvent-accessible according to the x-ray structure of porin OmpF.We then extended this method to probe for voltage gating of porin OmpF under different conditioned circumstances to assess the robustness of its application in complicated biological systems. Here E. coli was stimulated first with either a low ionic strength solution or a low pH buffer after harvest and exposed to OH radicals generated by Fenton reagents in solution for 3 min. Our data showed that the extent of oxidation of two polypeptides from extracellular loops remained constant independent of conditions that induced either channel closing or opening. However, we observed that oxidation level of peptides in β-stranded areas deep inside the porin channel was reduced when the environment changed from one that induced channel opening to another that induced channel closing. These data not only supported the voltage gating of porin OmpF in vivo but also revealed the molecular basis underlying voltage gating of porin OmpF.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.