Página 1 dos resultados de 154 itens digitais encontrados em 0.013 segundos

Orientation Determination of Interfacial β-sheet Structures in Situ

Nguyen, Khoi Tan; King, John Thomas; Chen, Zhan
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/07/2010 Português
Relevância na Pesquisa
58.475713%
Structural information such as orientations of interfacial proteins and peptides is important for understanding properties and functions of such biological molecules, which play crucial roles in biological applications and processes such as antimicrobial selectivity, membrane protein activity, biocompatibility, and biosensing performance. The α-helical and β-sheet structures are the most widely encountered secondary structures in peptides and proteins. In this paper, for the first time, a method to quantify the orientation of the interfacial β-sheet structure using a combined Attenuated Total Reflectance Fourier Transformation Infrared Spectroscopic (ATR-FTIR) and Sum Frequency Generation (SFG) vibrational spectroscopic study was developed. As an illustration of the methodology, the orientation of tachyplesin I, a 17-amino acid peptide with an anti-parallel β-sheet, adsorbed to polymer surfaces as well as associated with a lipid bilayer was determined using the regular and chiral SFG spectra, together with polarized ATR-FTIR amide I signals. Both the tilt angle (θ) and the twist angle (ψ) of the β-sheet at interfaces are determined. The developed method in this paper can be used to obtain in situ structural information of β-sheet components in complex molecules. The combination of this method and the existing methodology that is currently used to investigate α-helical structures will greatly broaden the application of optical spectroscopy in physical chemistry...

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel*

Xu, Xinping; Vysotskaya, Zhanna V.; Liu, Qinglian; Zhou, Lei
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
68.96998%
Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover...

The Proximal Hydrogen Bond Network Modulates Bacillus subtilis Nitric-oxide Synthase Electronic and Structural Properties

Brunel, Albane; Wilson, Adjélé; Henry, Laura; Dorlet, Pierre; Santolini, Jérôme
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
78.35888%
Bacterial nitric-oxide synthase (NOS)-like proteins are believed to be genuine NOSs. As for cytochromes P450 (CYPs), NOS-proximal ligand is a thiolate that exerts a push effect crucial for the process of dioxygen activation. Unlike CYPs, this catalytic electron donation seems controlled by a hydrogen bond (H-bond) interaction between the thiolate ligand and a vicinal tryptophan. Variations of the strength of this H-bond could provide a direct way to tune the stability along with the electronic and structural properties of NOS. We generated five different mutations of bsNOS Trp66, which can modulate this proximal H-bond. We investigated the effects of these mutations on different NOS complexes (FeIII, FeIICO, and FeIINO), using a combination of UV-visible absorption, EPR, FTIR, and resonance Raman spectroscopies. Our results indicate that (i) the proximal H-bond modulation can selectively decrease or increase the electron donating properties of the proximal thiolate, (ii) this modulation controls the σ-competition between distal and proximal ligands, (iii) this H-bond controls the stability of various NOS intermediates, and (iv) a fine tuning of the electron donation by the proximal ligand is required to allow at the same time oxygen activation and to prevent uncoupling reactions.

Switch I Closure Simultaneously Promotes Strong Binding to Actin and ADP in Smooth Muscle Myosin*

Decarreau, Justin A.; James, Nicholas G.; Chrin, Lynn R.; Berger, Christopher L.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
58.36063%
The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus*♦

Javaherian, Anoosh D.; Yusifov, Taleh; Pantazis, Antonios; Franklin, Sarah; Gandhi, Chris S.; Olcese, Riccardo
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
58.47695%
Large-conductance voltage- and Ca2+-dependent K+ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca2+ homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca2+ and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca2+- and Mg2+-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba2+-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca2+-binding site (the “calcium bowl”) reduced the Ca2+ and abolished the Mg2+ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca2+-binding site and that Mg2+ can bind to the calcium bowl with less affinity than Ca2+. Dynamic light scattering analysis revealed a reversible Ca2+-dependent decrease of the hydrodynamic radius of the gating ring...

Structure of a Pheromone Receptor-Associated MHC Molecule with an Open and Empty Groove

Olson, Rich; Huey-Tubman, Kathryn E; Dulac, Catherine; Bjorkman, Pamela J.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
58.4637%
Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide–binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I–binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8–10-mer class I–binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC–binding peptides...

The effects of manipulation of sedimentary iron and organic matter on sediment biogeochemistry and seagrasses in a subtropical carbonate environment

Ruiz-Halpern, Sergio; Macko, Stephen A.; Fourqurean, James W.
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
78.14371%
The microbial metabolism of organic matter (OM) in seagrass beds can create sulfidic conditions detrimental to seagrass growth; iron (Fe) potentially has ameliorating effects through titration of the sulfides and the precipitation of iron-sulfide minerals into the sediment. In this study, the biogeochemical effects of Fe availability and its interplay with sulfur and OM on sulfide toxicity, phosphorous (P) availability, seagrass growth and community structure were tested. The availability of Fe and OM was manipulated in a 2 × 2 factorial experiment arranged in a Latin square, with four replicates per treatment. The treatments included the addition of Fe, the addition of OM, the addition of both Fe and OM as well as no addition. The experiment was conducted in an oligotrophic, iron-deficient seagrass bed. Fe had an 84.5% retention efficiency in the sediments with the concentration of Fe increasing in the seagrass leaves over the course of the experiment. Porewater chemistry was significantly altered with a dramatic decrease in sulfide levels in Fe addition plots while sulfide levels increased in the OM addition treatments. Phosphorus increased in seagrass leaves collected in the Fe addition plots. Decreased sulfide stress was evidenced by heavier δ34S in leaves and rhizomes from plots to which Fe was added. The OM addition negatively affected seagrass growth but increased P availability; the reduced sulfide stress in Fe added plots resulted in elevated productivity. Fe availability may be an important determinant of the impact that OM has on seagrass vitality in carbonate sediments vegetated with seagrasses.

Roles of DNA Base Excision Repair in Maintaining the Integrity of DNA Methylation

Zhou, Jing
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
68.139844%
DNA methylation and demethylation are involved in regulation of gene expression. CpG clusters have been identified as hotspots of oxidative damages and mutagenesis. DNA base excision repair can remove oxidative DNA damage on CpG clusters and mediate an active DNA demethylation pathway. In this study, we examined the molecular mechanisms underlying interactions among DNA methylation, demethylation and BER. Our results demonstrated that a single 5-methylcytosine did not exhibit a significant effect on BER. Surprisingly we found that the abasic site completely inhibited the activity of thymine DNA glycosylase (TDG) leading to the sustainment of the mismatch efficiently extended by pol β. Interestingly, APE1 3’-5’ exonuclease could removed the mismatch. Our results demonstrate a molecular mechanisms underlying DNA base lesion and BER in maintenance of a normal DNA methylation pattern and a critical role of APE1 to combat pol β extension of the mismatch thereby reducing the introduction of mutagenesis.

Design and Synthesis of S-ribosylhomocysteine Analogues

Chbib, Christiane
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
78.133823%
Bacteria are known to release a large variety of small molecules known as autoinducers (AI) which effect quorum sensing (QS) initiation. The interruption of QS effects bacterial communication, growth and virulence. Three novel classes of S-ribosylhomocysteine (SRH) analogues as potential inhibitors of S-ribosylhomocysteinase (LuxS enzyme) and AI-2 modulators of QS were developed. The synthesis of 2-deoxy-2-bromo-SRH analogues was attempted by coupling of the corresponding 2-bromo-2-deoxypentafuranosyl precursors with the homocysteinate anion. The displacement of the bromide from C2 rather than the expected substitution of the mesylate from C5 was observed. The synthesis of 4-C-alkyl/aryl-S-ribosylhomocysteine analogues involved the following steps: (i) conversion of the D-ribose to the ribitol-4-ulose; (ii) diastereoselective addition of various alkyl or aryl or vinyl Grignard reagents to 4-ketone intermediate; (iii) oxidation of the primary hydroxyl group at C1 followed by the intramolecular ring closure to the corresponding 4-C-alkyl/aryl-substituted ribono-1,4-lactones; (iv) displacement of the activated 5-hydroxyl group with the protected homocysteinate. Treatment of the 4-C-alkyl/aryl-substituted SRH analogues with lithium triethylborohydride effected reduction of the ribonolactone to the ribose (hemiacetal) and subsequent global deprotection with trifluoroacetic acid provided 4-C-alkyl/aryl-SRHs. The 4-[thia]-SRH were prepared from the 1-deoxy-4-thioribose through the coupling of the α-fluoro thioethers (thioribosyl fluorides) with homocysteinate anion. The 4-[thia]-SRH analogues showed concentration dependent effect on the growth on las (50% inhibitory effect at 200 µg/mL). The most active was 1-deoxy-4-[thia]-SRH analogue with sufur atom in the ring oxidized to sulfoxide decreasing las gene activity to approximately 35% without affecting rhl gene. Neither of the tested compounds had effect on bioluminescence nor on total growth of V. harveyi...

Characterization of Juvenile Hormone Biosynthetic Enzymes in the Mosquito, Aedes aegypti

Nyati, Pratik
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
68.36688%
The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. They are synthesized and secreted by a pair of small endocrine glands, the corpora allata (CA), which are intimately connected to the brain. The enzymes involved in the biosynthesis of JH are attractive targets for the control of mosquito populations. This dissertation is a comprehensive functional study of five Aedes aegypti CA enzymes, HMG-CoA synthase (AaHMGS), mevalonate kinase (AaMK), phosphomevalonate kinase (AaPMK), farnesyl diphosphate synthase (AaFPPS) and farnesyl pyrophosphate phosphatase (AaFPPase). The enzyme AaHMGS catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce HMG-CoA. The enzyme does not require any co-factor, although its activity is enhanced by addition of Mg2+. The enzyme AaMK is a class I mevalonate kinase that catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate. Activity of AaMK is inhibited by isoprenoids. The enzyme AaPMK catalyzes the cation-dependent reversible reaction of phosphomevalonate and ATP to form diphosphate mevalonate and ADP. The enzyme AaFPPS catalyzes the condensation of isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP). The enzyme AaFPPS shows an unusual product regulation mechanism...

Mechanisms of Arsenic Detoxification and Resistance

Pillai, Jitesh Kannan
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
79.385303%
Arsenic is a ubiquitous environmental toxic substance. As a consequence of continual exposure to arsenic, nearly every organism, from Escherichia coli to humans have evolved arsenic detoxification pathways. One of the pathways is extrusion of arsenic from inside the cells, thereby conferring resistance. The R773 arsRDABC operon in E. coli encodes an ArsAB efflux pump that confers resistance to arsenite. ArsA is the catalytic subunit of the pump, while ArsB forms the oxyanion conducting pathway. ArsD is an arsenite metallochaperone that binds arsenite and transfers it to ArsA. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. The interaction between ArsA ATPase and ArsD metallochaperone was examined. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. Mutations in ArsA that propagate changes in hydrogen bonding and salt bridges to the ArsA-ArsD interface also affect their interactions. The second objective was to examine the mechanism of arsenite resistance through methylation and subsequent volatilization. Microbial ArsM (As(III) S-adenosylmethyltransferase) catalyzes the formation of trimethylarsine as the volatile end product. The net result is loss of arsenic from cells. The gene for CrArsM from the eukaryotic green alga Chlamydomonas reinhardtii was chemically synthesized and expressed in E. coli. The purified protein catalyzed the methylation of arsenite into methyl-...

Conformational Kinetics Study of MP-11 Using TIMS-MS and Molecular Dynamics

Harrilal, Christopher; Miksovska, Jaroslava; Schenk, Emily R
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
78.141963%
In the present work, we studied the conformational kinetics of microperoxidase 11 (MP-11), a digest fragment of cytochrome C that contains 11 amino acids with a covalently attached heme group. In particular, a novel technique recently developed at FIU in collaboration with Bruker Daltonics Inc. combined with theoretical calculation was used for the characterization of MP- 11 conformational space [1-3]. Accurate ion-neutral collision cross sections were measured for all MP-11 generated charge states. Since MP-11 (like cytochrome C) undergoes conformational changes as a function of the solvent state, MP-11 ions were produced by electrospray ionization (ESI) in order to preserve the initial solution state structure and analyzed on the basis of size-to-charge, inside the Trapped Ion Mobility Spectrometer (TIMS) followed by mass identification using a time-of-flight mass analyzer (MS) [4-5]. TIMS-MS has the advantage that molecular ions can be trapped for several seconds which allow us to study the kinetics and stability of various isomers as a function of time, initial pH value (6.1, 4.5, 3.1), and molecular ion temperature. Results showed that MP-11 conformations vary with pH levels and trapping time, and multiple interconversion pathways were observed for [M+2H]+2 and [M+3H]+3 charge states. Candidate structures were proposed for each conformation observed and main molecular interactions responsible for the conformational changes are discussed.

A Rapid and Sensitive High – Throughput Screening Method to Identify Compounds Targeting Protein-Nucleic Acids Interactions

Alonso, Nicole; Guillen, Roboan
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
78.133823%
DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian highmobility- group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

A Rapid and Sensitive High-Throughput Screening Method to Identify Compounds Targeting Protein–Nucleic Acids Interactions

Alonso, Nicole; Guillen, Roboan
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
78.133823%
DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian highmobility- group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

Linking Old Librarianship to New: Aligning 5-Steps of The Innovator's DNA in Creating Thematic Discovery Systems for the Everglades

Cooper, L. Bryan; Perez Martinez, Margarita
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
68.83885%
This poster presentation from the May 2015 Florida Library Association Conference, along with the Everglades Explorer discovery portal at http://ee.fiu.edu, demonstrates how traditional bibliographic and curatorial principles can be applied to: 1) selection, cross-walking and aggregation of metadata linking end-users to wide-spread digital resources from multiple silos; 2) harvesting of select PDFs, HTML and media for web archiving and access; 3) selection of CMS domains, sub-domains and folders for targeted searching using an API. Choosing content for this discovery portal is comparable to past scholarly practice of creating and publishing subject bibliographies, except metadata and data are housed in relational databases. This new and yet traditional capacity coincides with: Growth of bibliographic utilities (MarcEdit); Evolution of open-source discovery systems (eXtensible Catalog); Development of target-capable web crawling and archiving systems (Archive-it); and specialized search APIs (Google). At the same time, historical and technical changes – specifically the increasing fluidity and re-purposing of syndicated metadata – make this possible. It equally stems from the expansion of freely accessible digitized legacy and born-digital resources. Innovation principles helped frame the process by which the thematic Everglades discovery portal was created at Florida International University. The path -- to providing for more effective searching and co-location of digital scientific...

Recognition and Specificity Determinants of the Human Cbx Chromodomains*

Kaustov, Lilia; Ouyang, Hui; Amaya, Maria; Lemak, Alexander; Nady, Nataliya; Duan, Shili; Wasney, Gregory A.; Li, Zhihong; Vedadi, Masoud; Schapira, Matthieu; Min, Jinrong; Arrowsmith, Cheryl H.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
58.363325%
The eight mammalian Cbx proteins are chromodomain-containing proteins involved in regulation of heterochromatin, gene expression, and developmental programs. They are evolutionarily related to the Drosophila HP1 (dHP1) and Pc (dPc) proteins that are key components of chromatin-associated complexes capable of recognizing repressive marks such as trimethylated Lys-9 and Lys-27, respectively, on histone H3. However, the binding specificity and function of the human homologs, Cbx1–8, remain unclear. To this end we employed structural, biophysical, and mutagenic approaches to characterize the molecular determinants of sequence contextual methyllysine binding to human Cbx1–8 proteins. Although all three human HP1 homologs (Cbx1, -3, -5) replicate the structural and binding features of their dHP counterparts, the five Pc homologs (Cbx2, -4, -6, -7, -8) bind with lower affinity to H3K9me3 or H3K27me3 peptides and are unable to distinguish between these two marks. Additionally, peptide permutation arrays revealed a greater sequence tolerance within the Pc family and suggest alternative nonhistone sequences as potential binding targets for this class of chromodomains. Our structures explain the divergence of peptide binding selectivity in the Pc subfamily and highlight previously unrecognized features of the chromodomain that influence binding and specificity.

A Molecular Switch between the Outer and the Inner Vestibules of the Voltage-gated Na+ Channel*

Zarrabi, Touran; Cervenka, Rene; Sandtner, Walter; Lukacs, Peter; Koenig, Xaver; Hilber, Karlheinz; Mille, Markus; Lipkind, Gregory M.; Fozzard, Harry A.; Todt, Hannes
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
68.137197%
Voltage-gated ion channels are transmembrane proteins that undergo complex conformational changes during their gating transitions. Both functional and structural data from K+ channels suggest that extracellular and intracellular parts of the pore communicate with each other via a trajectory of interacting amino acids. No crystal structures are available for voltage-gated Na+ channels, but functional data suggest a similar intramolecular communication involving the inner and outer vestibules. However, the mechanism of such communication is unknown. Here, we report that amino acid Ile-1575 in the middle of transmembrane segment 6 of domain IV (DIV-S6) in the adult rat skeletal muscle isoform of the voltage-gated sodium channel (rNaV1.4) may act as molecular switch allowing for interaction between outer and inner vestibules. Cysteine scanning mutagenesis of the internal part of DIV-S6 revealed that only mutations at site 1575 rescued the channel from a unique kinetic state (“ultra-slow inactivation,” IUS) produced by the mutation K1237E in the selectivity filter. A similar effect was seen with I1575A. Previously, we reported that conformational changes of both the internal and the external vestibule are involved in the generation of IUS. The fact that mutations at site 1575 modulate IUS produced by K1237E strongly suggests an interaction between these sites. Our data confirm a previously published molecular model in which Ile-1575 of DIV-S6 is in close proximity to Lys-1237 of the selectivity filter. Furthermore...

Timothy Mutation Disrupts the Link between Activation and Inactivation in CaV1.2 Protein*

Depil, Katrin; Beyl, Stanislav; Stary-Weinzinger, Anna; Hohaus, Annette; Timin, Eugen; Hering, Steffen
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
58.362085%
The Timothy syndrome mutations G402S and G406R abolish inactivation of CaV1.2 and cause multiorgan dysfunction and lethal arrhythmias. To gain insights into the consequences of the G402S mutation on structure and function of the channel, we systematically mutated the corresponding Gly-432 of the rabbit channel and applied homology modeling. All mutations of Gly-432 (G432A/M/N/V/W) diminished channel inactivation. Homology modeling revealed that Gly-432 forms part of a highly conserved structure motif (G/A/G/A) of small residues in homologous positions of all four domains (Gly-432 (IS6), Ala-780 (IIS6), Gly-1193 (IIIS6), Ala-1503 (IVS6)). Corresponding mutations in domains II, III, and IV induced, in contrast, parallel shifts of activation and inactivation curves indicating a preserved coupling between both processes. Disruption between coupling of activation and inactivation was specific for mutations of Gly-432 in domain I. Mutations of Gly-432 removed inactivation irrespective of the changes in activation. In all four domains residues G/A/G/A are in close contact with larger bulky amino acids from neighboring S6 helices. These interactions apparently provide adhesion points, thereby tightly sealing the activation gate of CaV1.2 in the closed state. Such a structural hypothesis is supported by changes in activation gating induced by mutations of the G/A/G/A residues. The structural implications for CaV1.2 activation and inactivation gating are discussed.

Discovery & Born-Digital Archiving: Open Source Systems for Preservation and Access

Cooper, L. Bryan; Perez Martinez, Margarita
Fonte: FIU Digital Commons Publicador: FIU Digital Commons
Tipo: Artigo de Revista Científica Formato: application/vnd.openxmlformats-officedocument.presentationml.presentation
Português
Relevância na Pesquisa
78.551475%
The Everglades Explorer (EE) portal at http://ee.fiu.edu continues to evolve with the addition of the Internet Archive's Archive-It, and future planned alignment with the Digital Public Library of America (DPLA). The reasons for the development of the portal continue to exist, as originally perceived four years ago. Adaptations to ongoing change and system testing continues, and the use of Archive-It has broad institutional potential beyond EE. Cross-walking skills continue to grown, and will benefit future syndication and discovery system integration. The metadata normalization and harmonization will help save time for the end-user. Preservation and access to learning and research resources for the Greater Everglades Watershed -- stretching from the Kissimmee River Basin to the Florida Keys -- can continue to expand.

NMR Structure Improvement: A Structural Bioinformatics & Visualization Approach

Block, Jeremy
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação Formato: 32676308 bytes; application/pdf
Publicado em //2010 Português
Relevância na Pesquisa
58.54031%

The overall goal of this project is to enhance the physical accuracy of individual models in macromolecular NMR (Nuclear Magnetic Resonance) structures and the realism of variation within NMR ensembles of models, while improving agreement with the experimental data. A secondary overall goal is to combine synergistically the best aspects of NMR and crystallographic methodologies to better illuminate the underlying joint molecular reality. This is accomplished by using the powerful method of all-atom contact analysis (describing detailed sterics between atoms, including hydrogens); new graphical representations and interactive tools in 3D and virtual reality; and structural bioinformatics approaches to the expanded and enhanced data now available.

The resulting better descriptions of macromolecular structure and its dynamic variation enhances the effectiveness of the many biomedical applications that depend on detailed molecular structure, such as mutational analysis, homology modeling, molecular simulations, protein design, and drug design.

; Dissertation