Cluster Of Amino Acids Research Articles

Chirality and Packing in Small Proline Clusters

The chiral composition of amino acid clusters may be related to the origin of chirality in biological systems. Here, we use ion mobility/mass spectrometry techniques to investigate the gas phase structures of singly charged proline clusters containing two to six monomers. Using deuterated L-proline (L(D7)) and different electrospray solution compositions varying from enantiopure (50:50 L:L(D7)) to racemic (50:50 L(D7):D), it is possible to study collision cross sections of L-, D-, and mixed xL:xD-proline clusters (where x refers to the number of monomers). These results show that [2Pro+H](+) and [3Pro+H](+) clusters, previously shown (Holliday et al. J. Phys. Chem. A 2012, DOI: 10.1021/jp302677n) to have a very small heterochiral preference, have similar collision cross sections for homochiral and heterochiral proline assemblies. The [4Pro+H](+) and [6Pro+H](+) clusters that exhibit homochiral preference have smaller collision cross sections for homochiral clusters and larger collision cross sections for heterochiral clusters. The [5Pro+H](+) cluster with heterochiral preference has a smaller collision cross section for its heterochiral compositions than for its homochiral compositions. These results suggest that the packing efficiency of subunits within each cluster influences the stability and prevalence of proline multimers as either homochiral or mixed L- and D- clusters.

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na+/H+ exchanger isoform 1)

NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations.

P107 Identification of adaptive mutations in the nucleoprotein of pandemic influenza A viruses required to evade restriction from the interferon induced MxA protein

Introduction The interferon induced human MxA protein is a potent restriction factor against avian influenza A virus infections. We therefore predicted that zoonotic transmission of these viruses into the human population is accompanied by the acquisition of adaptive mutations allowing evasion from MxA restriction. Methods The H5N1 polymerase reconstitution assay was utilized to identify adaptive mutations in the nucleoproteins (NP) of pandemic viruses required for resistance against human MxA. Highly pathogenic H5N1 viruses harboring the identified adaptive mutations in NP were generated and their growth properties were determined in cell culture. The evolutionary pressure to maintain the MxA resistance-contributing amino acids was assessed using the NCBI Influenza Virus Sequence Database. Results We identified the MxA-adaptive mutations in the nucleoprotein (NP) of the pandemic strains A/Brevig Mission/1/1918(H1N1) (1918) and A/Hamburg/4/2009(H1N1) (pH1N1). Intriguingly, the amino acids conferring resistance towards MxA differ in both strains, but cluster to the same area of the body domain of NP. Sequence analysis revealed that the amino acid cluster required for MxA resistance in the 1918 strain remained highly conserved in all descendant seasonal and pandemic strains. However, the amino acid cluster in NP of the pH1N1 strain contains three unprecedented adaptive mutations, indicating an independent evolution of MxA resistance in this porcine-derived virus. Introduction of either the 1918 or the pH1N1 amino acid cluster into NP of an H5N1 virus mediated resistance to MxA but also decreased viral fitness. Vice versa, mutation of the corresponding amino acid cluster in pH1N1 NP to avian signature impaired MxA resistance, while viral growth was increased. Conclusion Taken together, the acquisitions of NP mutations required for MxA resistance emerged in both the 1918 and the 2009 pandemic strain independently and were most likely accompanied by compensatory mutations to overcome the associated strong attenuation.

Probing a Polar Cluster in the Retinal Binding Pocket of Bacteriorhodopsin by a Chemical Design Approach

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C13 of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.

Peptide-specific Transfer of N-Acetylgalactosamine to O-Linked Glycans by the Glycosyltransferases β1,4-N-Acetylgalactosaminyl Transferase 3 (β4GalNAc-T3) and β4GalNAc-T4

N- and O-linked oligosaccharides on pro-opiomelanocortin both bear the unique terminal sequence SO(4)-4-GalNAcβ1,4GlcNAcβ. We previously demonstrated that protein-specific transfer of GalNAc to N-linked oligosaccharides on glycoprotein substrates is dependent on the presence of both an oligosaccharide acceptor and a peptide recognition motif consisting of a cluster of basic amino acids. We characterized how two β1,4-N-acetylgalactosaminyltransferases, β4GalNAc-T3 and β4GalNAc-T4, require the presence of both the peptide recognition motif and the N-linked oligosaccharide acceptors to transfer GalNAc in β1,4-linkage to GlcNAc in vivo and in vitro. We now show that β4GalNAc-T3 and β4GalNAc-T4 are able to utilize the same peptide motif to selectively add GalNAc to β1,6-linked GlcNAc in core 2 O-linked oligosaccharide structures to form Galβ1,3(GalNAcβ1,4GlcNAcβ1,6)GalNAcαSer/Thr. The β1,4-linked GalNAc can be further modified with 4-linked sulfate by either GalNAc-4-sulfotransferase 1 (GalNAc-4-ST1) (CHST8) or GalNAc-4-ST2 (CHST9) or with α2,6-linked N-acetylneuraminic acid by α2,6-sialyltransferase 1 (ST6Gal1), thus generating a family of unique GalNAcβ1,4GlcNAcβ (LacdiNAc)-containing structures on specific glycoproteins.

Inhibition of Human Respiratory Syncytial Virus Infectivity by a Dendrimeric Heparan Sulfate-Binding Peptide

Respiratory syncytial virus (RSV) interacts with cell surface heparan sulfate proteoglycans (HSPGs) to initiate infection. The interaction of RSV with HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In the present study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was screened with the aim of identifying peptides able to bind HSPGs and thus block RSV attachment and infectivity. Of the compounds identified, the dendrimer SB105-A10 was the most potent inhibitor of RSV infectivity, with 50% inhibitory concentrations (IC(50)s) of 0.35 μM and 0.25 μM measured in Hep-2 and A549 cells, respectively. SB105-A10 was found to bind to both cell types via HSPGs, suggesting that its antiviral activity is indeed exerted by competing with RSV for binding to cell surface HSPGs. SB105-A10 prevented RSV infection when added before the viral inoculum, in line with its proposed HSPG-binding mechanism of action; moreover, antiviral activity was also exhibited when SB105-A10 was added postinfection, as it was able to reduce the cell-to-cell spread of the virus. The antiviral potential of SB105-A10 was further assessed using human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. SB105-A10 strongly reduced RSV infectivity in this model and exhibited no signs of cytotoxicity or proinflammatory effects. Together, these features render SB105-A10 an attractive candidate for further development as a RSV inhibitor to be administered by aerosol delivery.

Reactive oxygen species affect ATP hydrolysis by targeting a highly conserved amino acid cluster in the thylakoid ATP synthase γ subunit

The vast majority of organisms produce ATP by a membrane-bound rotating protein complex, termed F-ATP synthase. In chloroplasts, the corresponding enzyme generates ATP by using a transmembrane proton gradient generated during photosynthesis, a process releasing high amounts of molecular oxygen as a natural byproduct. Due to its chemical properties, oxygen can be reduced incompletely which generates several highly reactive oxygen species (ROS) that are able to oxidize a broad range of biomolecules. In extension to previous studies it could be shown that ROS dramatically decreased ATP synthesis in situ and affected the CF1 portion in vitro. A conserved cluster of three methionines and a cysteine on the chloroplast γ subunit could be identified by mass spectrometry to be oxidized by ROS. Analysis of amino acid substitutions in a hybrid F1 assembly system indicated that these residues were exclusive catalytic targets for hydrogen peroxide and singlet oxygen, although it could be deduced that additional unknown amino acid targets might be involved in the latter reaction. The cluster was tightly integrated in catalytic turnover since mutants varied in MgATPase rates, stimulation by sulfite and chloroplast-specific γ subunit redox-modulation. Some partial disruptions of the cluster by mutagenesis were dominant over others regarding their effects on catalysis and response to ROS.

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.

Monoclonal antibodies to the V2 domain of MN-rgp120: fine mapping of epitopes and inhibition of α4β7 binding.

BackgroundRecombinant gp120 (MN-rgp120) was a major component of the AIDSVAX B/E vaccine used in the RV144 trial. This was the first clinical trial to show that vaccination could prevent HIV infection in humans. A recent RV144 correlates of protection study found that protection correlated with the presence of antibodies to the V2 domain. It has been proposed that antibodies to the α4β7 binding site in the V2 domain might prevent HIV-1 infection by blocking the ability of virions to recognize α4β7 on activated T-cells. In this study we investigated the specificity of monoclonal antibodies (MAbs) to the V2 domain of MN-rgp120 and examined the possibility that these antibodies could inhibit the binding of MN-rgp120 to the α4β7 integrin.Methodology/Principal FindingsNine MAbs to the V2 domain were isolated from mice immunized with recombinant envelope proteins. The ability of these MAbs to inhibit HIV infection, block the binding of gp120 to CD4, and block the binding of MN-rgp120 to the α4β7 integrin was measured. Mutational analysis showed that eight of the MAbs recognized two immunodominant clusters of amino acids (166–168 and 178–183) located at either end of the C strand within the four-strand anti-parallel sheet structure comprising the V1/V2 domain.Conclusions/SignificanceThese studies showed that the antigenic structure of the V2 domain is exceedingly complex and that MAbs isolated from mice immunized with MN-rgp120 exhibited a high level of strain specificity compared to MAbs to the V2 domain isolated from HIV-infected humans. We found that immunization with MN-rgp120 readily elicits antibodies to the V2 domain and some of these were able to block the binding of MN-rgp120 to the α4β7 integrin.

Structural basis for homeodomain recognition by the cell-cycle regulator Geminin

Homeodomain-containing transcription factors play a fundamental role in the regulation of numerous developmental and cellular processes. Their multiple regulatory functions are accomplished through context-dependent inputs of target DNA sequences and collaborating protein partners. Previous studies have well established the sequence-specific DNA binding to homeodomains; however, little is known about how protein partners regulate their functions through targeting homeodomains. Here we report the solution structure of the Hox homeodomain in complex with the cell-cycle regulator, Geminin, which inhibits Hox transcriptional activity and enrolls Hox in cell proliferative control. Side-chain carboxylates of glutamates and aspartates in the C terminus of Geminin generate an overall charge pattern resembling the DNA phosphate backbone. These residues provide electrostatic interactions with homeodomain, which combine with the van der Waals contacts to form the stereospecific complex. We further showed that the interaction with Geminin is homeodomain subclass-selective and Hox paralog-specific, which relies on the stapling role of residues R43 and M54 in helix III and the basic amino acid cluster in the N terminus. Interestingly, we found that the C-terminal residue Ser184 of Geminin could be phosphorylated by Casein kinase II, resulting in the enhanced binding to Hox and more potent inhibitory effect on Hox transcriptional activity, indicating an additional layer of regulation. This structure provides insight into the molecular mechanism underlying homeodomain-protein recognition and may serve as a paradigm for interactions between homeodomains and DNA-competitive peptide inhibitors.

Identification of multiply charged proteins and amino acid clusters by liquid nitrogen assisted spray ionization mass spectrometry

The development of liquid nitrogen assisted spray ionization mass spectrometry (LNASI MS) for the analysis of multiply charged proteins (insulin, ubiquitin, cytochrome c, α-lactalbumin, myoglobin and BSA), peptides (glutathione, HW6, angiotensin-II and valinomycin) and amino acid (arginine) clusters is described. The charged droplets are formed by liquid nitrogen assisted sample spray through a stainless steel nebulizer and transported into mass analyzer for the identification of multiply charged protein ions. The effects of acids and modifier volumes for the efficient ionization of the above analytes in LNASI MS were carefully investigated. Multiply charged proteins and amino acid clusters were effectively identified by LNASI MS. The present approach can effectively detect the multiply charged states of cytochrome c at 400nM. A comparison between LNASI and ESI, CSI, SSI and V-EASI methods on instrumental conditions, applied temperature and observed charge states for the multiply charged proteins, shows that the LNASI method produces the good quality spectra of amino acid clusters at ambient conditions without applied any electric field and heat. To date, we believe that the LNASI method is the most simple, low cost and provided an alternative paradigm for production of multiply charged ions by LNASI MS, just as ESI-like ions yet no need for applying any electrical field and it could be operated at low temperature for generation of highly charged protein/peptide ions.

Structure-Guided Topographic Mapping and Mutagenesis to Elucidate Binding Sites for the Human Ether-a-Go-Go-Related Gene 1 Potassium Channel (KCNH2) Activator NS1643

Loss-of -function mutations in human ether-a-go-go-related gene 1 (hERG1) is associated with life-threatening arrhythmias. hERG1 activators are being developed as treatments for acquired or genetic forms of long QT syndrome. The locations of the putative binding pockets for activators are still being elucidated. In silico docking of the activator 1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea (NS1643) to an S1-S6 transmembrane homology model of hERG1 predicted putative binding sites. The predictions of the in silico docking guided subsequent in vitro mutagenesis and electrophysiological measurements. The novel interacting site for NS1643 is predicted around Asn629 at the outer mouth of the channel. The applied N629H mutation is the sole amino acid replacement in the literature that abrogates the NS1643-induced left shift of the V(1/2) of activation. In contrast, both N629T and N629D showed pharmacologic responses similar to wild type. Another important interacting pocket is predicted at the intracellular surface in the S4-S5 linker. Mutagenesis of the residues critical to interactions in this pocket had major effects on the pharmacologic response to NS1643. The inward conductance elicited by hyperpolarization of D540K hERG1 was abrogated by NS1643 treatment, suggesting that it alters the inward movement of the S4 segment. The neighboring E544L mutation markedly exaggerated tail-current responses to NS1643. However, an L564A substitution inhibited drug response. Structure-guided mutagenesis identified widespread clusters of amino acids modulating drug-induced shifts in inactivation; such modulation may reflect allosteric changes in tertiary structure. Model-guided mutagenesis led to the discovery of a range of novel interacting residues that modify NS1643-induced pharmacologic responses.

LAHEDES: the LAGLIDADG homing endonuclease database and engineering server

LAGLIDADG homing endonucleases (LHEs) are DNA cleaving enzymes, also termed ‘meganucleases’ that are employed as gene-targeting reagents. This use of LHEs requires that their DNA specificity be altered to match sequences in genomic targets. The choice of the most appropriate LHE to target a particular gene is facilitated by the growing number of such enzymes with well-characterized activities and structures. ‘LAHEDES’ (The LAGLIDADG Homing Endonuclease Database and Engineering Server) provides both an online archive of LHEs with validated DNA cleavage specificities and DNA-binding interactions, as well as a tool for the identification of DNA sequences that might be targeted by various LHEs. Searches can be performed using four separate scoring algorithms and user-defined choices of LHE scaffolds. The webserver subsequently provides information regarding clusters of amino acids that should be interrogated during engineering and selection experiments. The webserver is fully open access and can be found at http://homingendonuclease.net.

Transglutaminase-2 Interaction with Heparin: IDENTIFICATION OF A HEPARIN BINDING SITE THAT REGULATES CELL ADHESION TO FIBRONECTIN-TRANSGLUTAMINASE-2 MATRIX

Heparan sulfate proteoglycans are critical binding partners for extracellular tranglutaminase-2 (TG2), a multifunctional protein involved in tissue remodeling events related to organ fibrosis and cancer progression. We previously showed that TG2 has a strong affinity for heparan sulfate (HS)/heparin and reported that the heparan sulfate proteoglycan syndecan-4 acts as a receptor for TG2 via its HS chains in two ways: by increasing TG2-cell surface trafficking/externalization and by mediating RGD-independent cell adhesion to fibronectin-TG2 matrix during wound healing. Here we have investigated the molecular basis of this interaction. Site-directed mutagenesis revealed that either mutation of basic RRWK (262-265) or KQKRK (598-602) clusters, forming accessible heparin binding sequences on the TG2 three-dimensional structure, led to an almost complete reduction of heparin binding, indicating that both clusters contribute to form a single binding surface. Mutation of residues Arg(19) and Arg(28) also led to a significant reduction in heparin binding, suggesting their involvement. Our findings indicate that the heparin binding sites on TG2 mainly comprise two clusters of basic amino acids, which are distant in the linear sequence but brought into spatial proximity in the folded "closed" protein, forming a high affinity heparin binding site. Molecular modeling showed that the identified site can make contact with a single heparin-derived pentasaccharide. The TG2-heparin binding mutants supported only weak RGD-independent cell adhesion compared with wild type TG2 or mutants with retained heparin binding, and both heparin binding clusters were critical for TG2-mediated cell adhesion. These findings significantly advance our knowledge of how HS/heparin influences the adhesive function of TG2.

Extended protein/water H-bond networks in photosynthetic water oxidation

Oxidation of water molecules in the photosystem II (PSII) protein complex proceeds at the manganese–calcium complex, which is buried deeply in the lumenal part of PSII. Understanding the PSII function requires knowledge of the intricate coupling between the water-oxidation chemistry and the dynamic proton management by the PSII protein matrix. Here we assess the structural basis for long-distance proton transfer in the interior of PSII and for proton management at its surface. Using the recent high-resolution crystal structure of PSII, we investigate prominent hydrogen-bonded networks of the lumenal side of PSII. This analysis leads to the identification of clusters of polar groups and hydrogen-bonded networks consisting of amino acid residues and water molecules. We suggest that long-distance proton transfer and conformational coupling is facilitated by hydrogen-bonded networks that often involve more than one protein subunit. Proton-storing Asp/Glu dyads, such as the D1-E65/D2-E312 dyad connected to a complex water-wire network, may be particularly important for coupling protonation states to the protein conformation. Clusters of carboxylic amino acids could participate in proton management at the lumenal surface of PSII. We propose that rather than having a classical hydrophobic protein interior, the lumenal side of PSII resembles a complex polyelectrolyte with evolutionary optimized hydrogen-bonding networks. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Predictive modelling of angiotensin converting enzyme inhibitory dipeptides

Abstract The ability of docking to predict angiotensin converting enzyme (ACE) inhibitory dipeptide sequences was assessed using AutoDock Vina. All potential dipeptides and phospho-dipeptides were docked and scored. Peptide intestinal stability was assessed using a prediction amino acid clustering model. Selected dipeptides, having AutoDock Vina scores ⩽ −8.1 and predicted to be ‘stable’ intestinally, were characterised, using LIGPLOT and for ACE-inhibitory potency. Two newly identified ACE-inhibitory dipeptides, Asp-Trp and Trp-Pro, having Vina scores of −8.3 and −8.6 gave IC 50 values of 258 ± 4.23 and 217 ± 15.7 μM, respectively. LIGPLOT analysis indicated no zinc interaction for these dipeptides. Phospho-dipeptides were predicted to have a good affinity for ACE. However, the experimentally determined IC 50 results did not correlate since, for example, Trp-pThr and Pro-pTyr, having Vina scores of −8.5 and −8.1, respectively, displayed IC 50 values of >500 μM. While docking allowed identification of new ACE inhibitory dipeptides, it may not be a fully reliable predictive tool in all cases.

A new protein graph model for function prediction

As several structural proteomic projects are producing an increasing number of protein structures with unknown function, methods that can reliably predict protein functions from protein structures are in urgent need. In this paper, we present a method to explore the clustering patterns of amino acids on the 3-dimensional space for protein function prediction. First, amino acid residues on a protein structure are clustered into spatial groups using hierarchical agglomerative clustering, based on the distance between them. Second, the protein structure is represented using a graph, where each node denotes a cluster of amino acids. The nodes are labeled with an evolutionary profile derived from the multiple alignment of homologous sequences. Then, a shortest-path graph kernel is used to calculate similarities between the graphs. Finally, a support vector machine using this graph kernel is used to train classifiers for protein function prediction. We applied the proposed method to two separate problems, namely, prediction of enzymes and prediction of DNA-binding proteins. In both cases, the results showed that the proposed method outperformed other state-of-the-art methods.

Collision-Induced Dissociation of Halide Ion–Arginine Complexes: Evidence for Anion-Induced Zwitterion Formation in Gas-Phase Arginine

We report the first low-energy collisional-induced dissociation studies of the X(-)·arginine (X(-) = F(-), Cl(-), Br(-), I(-), NO(3)(-), ClO(3)(-)) series of clusters to investigate the novel phenomenom of anion-induced zwitterion formation in a gas-phase amino acid. Fragmentation of the small halide ion clusters (F(-)·arginine and Cl(-)·arginine) is dominated by deprotonation of the arginine, whereas the major fragmentation channel for the largest ion clusters (I(-)·arginine and ClO(3)(-)·arginine) corresponds to simple cluster fission into the ion and neutral molecule. However, the fragmentation profiles of Br(-)·arginine and NO(3)(-)·arginine, display distinctive features that are consistent with the presence of the zwitterionic form of the amino acid in these clusters. The various dissociation pathways have been studied as a function of % collision energy and are discussed in comparison to the fragmentation profiles of protonated and deprotonated arginine. Electronic structure calculations are presented for Br(-)·arginine to support the presence of the zwitterionic amino acid in this complex. The results obtained in this work provide important information on the low-energy potential energy surfaces of these anion-amino acid clusters and reveal the presence of several overlapping surfaces in the low-energy region for the Br(-)·arginine and NO(3)(-)·arginine systems.

Nuclear import of hTERT requires a bipartite nuclear localization signal mediated by Akt phosphorylation

Sustained cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity and protection. Although nuclear import of telomerase transcriptase (hTERT) is required for telomerase activity to elongate telomeres in vivo, the molecular mechanism regulating nuclear localization of hTERT is unclear. We have identified a bipartite nuclear localization signal (NLS; amino acid residues 222-240) that is responsible for nuclear import of hTERT. Immunofluorescence imaging of hTERT revealed that mutations in any of the bipartite NLS sequences result in decreased nuclear fluorescence intensity compared with wild-type hTERT. We also show that Akt-mediated phosphorylation at serine 227 is necessary for directing nuclear translocation of hTERT. Interestingly, serine 227 is located between two clusters of basic amino acids in the bipartite NLS. Inactivation of Akt activity by a dominant-negative mutant or wortmannin treatment attenuated nuclear localization of hTERT. We further show that both bipartite NLS and serine 227 in hTERT are required for cell immortalization of normal human foreskin fibroblast cells. Taken together, our findings reveal a previously unknown regulatory mechanism for nuclear import of hTERT through a bipartite NLS mediated by Akt phosphorylation, which represents an alternative pathway for modulating telomerase activity in cancer.