共查询到20条相似文献,搜索用时 46 毫秒
1.
Oliver Fürst Colin?G. Nichols Guillaume Lamoureux Nazzareno D’Avanzo 《Biophysical journal》2014,107(12):2786-2796
Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket. 相似文献
2.
Assembly and Kinetic Folding Pathways of a Tetrameric β-Sheet Complex: Molecular Dynamics Simulations on Simplified Off-Lattice Protein Models 下载免费PDF全文
We have performed discontinuous molecular dynamic simulations of the assembly and folding kinetics of a tetrameric β-sheet complex that contains four identical four-stranded antiparallel β-sheet peptides. The potential used in the simulation is a hybrid Go-type potential characterized by the bias gap parameter g, an artificial measure of a model protein's preference for its native state, and the intermolecular contact parameter η, which measures the ratio of intermolecular to intramolecular native attractions. The formation of the β-sheet complex and its equilibrium properties strongly depend on the size of the intermolecular contact parameter η. The ordered β-sheet complex in the folded state and nonaligned β-sheets or tangled chains in the misfolded state are distinguished by measuring the squared radius of gyration and the fraction of native contacts Q. The folding yield for the folded state is high at intermediate values of η, but is low at both small and large values of η. The folded state at small η is liquid-like, but is solid-like at both intermediate and large η. The misfolded state at small η contains nonaligned β-sheets and tangled chains with poor secondary structure at large η. Various folding pathways via dimeric and trimeric intermediates are observed, depending on η. Comparison with experimental results on protein aggregation indicates that intermediate η values are most appropriate for modeling fibril formation and small η values are most appropriate for modeling the formation of amorphous aggregates. 相似文献
3.
Staphylococcus aureus expresses numerous virulence factors that aid in immune evasion. The four-domain staphylococcal immunoglobulin binding (Sbi) protein interacts with complement component 3 (C3) and its thioester domain (C3d)-containing fragments. Recent structural data suggested two possible modes of binding of Sbi domain IV (Sbi-IV) to C3d, but the physiological binding mode remains unclear. We used a computational approach to provide insight into the C3d-Sbi-IV interaction. Molecular dynamics (MD) simulations showed that the first binding mode (PDB code 2WY8) is more robust than the second (PDB code 2WY7), with more persistent polar and nonpolar interactions, as well as conserved interfacial solvent accessible surface area. Brownian dynamics and steered MD simulations revealed that the first binding mode has faster association kinetics and maintains more stable intermolecular interactions compared to the second binding mode. In light of available experimental and structural data, our data confirm that the first binding mode represents Sbi-IV interaction with C3d (and C3) in a physiological context. Although the second binding mode is inherently less stable, we suggest a possible physiological role. Both binding sites may serve as a template for structure-based design of novel complement therapeutics. 相似文献
4.
Maria?Grazia Ortore Joci?N.A. Macedo Ana?Paula?U. Araujo Claudio Ferrero Paolo Mariani Francesco Spinozzi Rosangela Itri 《Biophysical journal》2015,108(12):2896-2902
Septins comprise a family of proteins involved in a variety of cellular processes and related to several human pathologies. They are constituted by three structural domains: the N- and C-terminal domains, highly variable in length and composition, and the central domain, involved in the guanine nucleotide (GTP) binding. Thirteen different human septins are known to form heterogeneous complexes or homofilaments, which are stabilized by specific interactions between the different interfaces present in the domains. In this work, we have investigated by in-solution small-angle x-ray scattering the structural and thermodynamic properties of a human septin 3 construct, SEPT3-GC, which contains both of both interfaces (G and NC) responsible for septin-septin interactions. In order to shed light on the role of these interactions, small-angle x-ray scattering measurements were performed in a wide range of temperatures, from 2 up to 56°C, both with and without a nonhydrolysable form of GTP (GTPγS). The acquired data show a temperature-dependent coexistence of monomers, dimers, and higher-order aggregates that were analyzed using a global fitting approach, taking into account the crystallographic structure of the recently reported SEPT3 dimer, PDB:3SOP. As a result, the enthalpy, entropy, and heat capacity variations that control the dimer-monomer dissociation equilibrium in solution were derived and GTPγS was detected to increase the enthalpic stability of the dimeric species. Moreover, a temperature increase was observed to induce dissociation of SEPT3-GC dimers into monomers just preceding their reassembling into amyloid aggregates, as revealed by the Thioflavin-T fluorescence assays. 相似文献
5.
Herman K. Edskes Hima J. Khamar Chia-Lin Winchester Alexandria J. Greenler Albert Zhou Ryan P. McGlinchey Anton Gorkovskiy Reed B. Wickner 《Genetics》2014,198(2):605-616
Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p’s from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae
Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function. 相似文献
6.
Hailey N. Conover Scott A. Lujan Mary J. Chapman Deborah A. Cornelio Rabab Sharif Jessica S. Williams Alan B. Clark Francheska Camilo Thomas A. Kunkel Juan Lucas Argueso 《Genetics》2015,201(3):951-961
We show by whole genome sequence analysis that loss of RNase H2 activity increases loss of heterozygosity (LOH) in Saccharomyces cerevisiae diploid strains harboring the pol2-M644G allele encoding a mutant version of DNA polymerase ε that increases ribonucleotide incorporation. This led us to analyze the effects of loss of RNase H2 on LOH and on nonallelic homologous recombination (NAHR) in mutant diploid strains with deletions of genes encoding RNase H2 subunits (rnh201Δ, rnh202Δ, and rnh203Δ), topoisomerase 1 (TOP1Δ), and/or carrying mutant alleles of DNA polymerases ε, α, and δ. We observed an ∼7-fold elevation of the LOH rate in RNase H2 mutants encoding wild-type DNA polymerases. Strains carrying the pol2-M644G allele displayed a 7-fold elevation in the LOH rate, and synergistic 23-fold elevation in combination with rnh201Δ. In comparison, strains carrying the pol2-M644L mutation that decreases ribonucleotide incorporation displayed lower LOH rates. The LOH rate was not elevated in strains carrying the pol1-L868M or pol3-L612M alleles that result in increased incorporation of ribonucleotides during DNA synthesis by polymerases α and δ, respectively. A similar trend was observed in an NAHR assay, albeit with smaller phenotypic differentials. The ribonucleotide-mediated increases in the LOH and NAHR rates were strongly dependent on TOP1. These data add to recent reports on the asymmetric mutagenicity of ribonucleotides caused by topoisomerase 1 processing of ribonucleotides incorporated during DNA replication. 相似文献
7.
Jenneke Leentjens Jessica Quintin Jelle Gerretsen Matthijs Kox Peter Pickkers Mihai G. Netea 《PloS one》2014,9(9)
Rationale
To prevent or combat infection, increasing the effectiveness of the immune response is highly desirable, especially in case of compromised immune system function. However, immunostimulatory therapies are scarce, expensive, and often have unwanted side-effects. β-glucans have been shown to exert immunostimulatory effects in vitro and in vivo in experimental animal models. Oral β-glucan is inexpensive and well-tolerated, and therefore may represent a promising immunostimulatory compound for human use.Methods
We performed a randomized open-label intervention pilot-study in 15 healthy male volunteers. Subjects were randomized to either the β -glucan (n = 10) or the control group (n = 5). Subjects in the β-glucan group ingested β-glucan 1000 mg once daily for 7 days. Blood was sampled at various time-points to determine β-glucan serum levels, perform ex vivo stimulation of leukocytes, and analyze microbicidal activity.Results
β-glucan was barely detectable in serum of volunteers at all time-points. Furthermore, neither cytokine production nor microbicidal activity of leukocytes were affected by orally administered β-glucan.Conclusion
The present study does not support the use of oral β-glucan to enhance innate immune responses in humans.Trial Registration
ClinicalTrials.gov NCT01727895相似文献8.
Erica Walkinshaw Yunchao Gai Caitlin Farkas Daniel Richter Eric Nicholas Krystyna Keleman Ronald L. Davis 《Genetics》2015,199(4):1173-1182
Genetic screens in Drosophila melanogaster and other organisms have been pursued to filter the genome for genetic functions important for memory formation. Such screens have employed primarily chemical or transposon-mediated mutagenesis and have identified numerous mutants including classical memory mutants, dunce and rutabaga. Here, we report the results of a large screen using panneuronal RNAi expression to identify additional genes critical for memory formation. We identified >500 genes that compromise memory when inhibited (low hits), either by disrupting the development and normal function of the adult animal or by participating in the neurophysiological mechanisms underlying memory formation. We also identified >40 genes that enhance memory when inhibited (high hits). The dunce gene was identified as one of the low hits and further experiments were performed to map the effects of the dunce RNAi to the α/β and γ mushroom body neurons. Additional behavioral experiments suggest that dunce knockdown in the mushroom body neurons impairs memory without significantly affecting acquisition. We also characterized one high hit, sickie, to show that RNAi knockdown of this gene enhances memory through effects in dopaminergic neurons without apparent effects on acquisition. These studies further our understanding of two genes involved in memory formation, provide a valuable list of genes that impair memory that may be important for understanding the neurophysiology of memory or neurodevelopmental disorders, and offer a new resource of memory suppressor genes that will aid in understanding restraint mechanisms employed by the brain to optimize resources. 相似文献
9.
DNA polymerases (Pols) ε and δ perform the bulk of yeast leading- and lagging-strand DNA synthesis. Both Pols possess intrinsic proofreading exonucleases that edit errors during polymerization. Rare errors that elude proofreading are extended into duplex DNA and excised by the mismatch repair (MMR) system. Strains that lack Pol proofreading or MMR exhibit a 10- to 100-fold increase in spontaneous mutation rate (mutator phenotype), and inactivation of both Pol δ proofreading (pol3-01) and MMR is lethal due to replication error-induced extinction (EEX). It is unclear whether a similar synthetic lethal relationship exists between defects in Pol ε proofreading (pol2-4) and MMR. Using a plasmid-shuffling strategy in haploid Saccharomyces cerevisiae, we observed synthetic lethality of pol2-4 with alleles that completely abrogate MMR (msh2Δ, mlh1Δ, msh3Δ msh6Δ, or pms1Δ mlh3Δ) but not with partial MMR loss (msh3Δ, msh6Δ, pms1Δ, or mlh3Δ), indicating that high levels of unrepaired Pol ε errors drive extinction. However, variants that escape this error-induced extinction (eex mutants) frequently emerged. Five percent of pol2-4 msh2Δ eex mutants encoded second-site changes in Pol ε that reduced the pol2-4 mutator phenotype between 3- and 23-fold. The remaining eex alleles were extragenic to pol2-4. The locations of antimutator amino-acid changes in Pol ε and their effects on mutation spectra suggest multiple mechanisms of mutator suppression. Our data indicate that unrepaired leading- and lagging-strand polymerase errors drive extinction within a few cell divisions and suggest that there are polymerase-specific pathways of mutator suppression. The prevalence of suppressors extragenic to the Pol ε gene suggests that factors in addition to proofreading and MMR influence leading-strand DNA replication fidelity. 相似文献
10.
11.
Understanding which peptides and proteins have the potential to undergo amyloid formation and what driving forces are responsible for amyloid-like fiber formation and stabilization remains limited. This is mainly because proteins that can undergo structural changes, which lead to amyloid formation, are quite diverse and share no obvious sequence or structural homology, despite the structural similarity found in the fibrils. To address these issues, a novel approach based on recursive feature selection and feed-forward neural networks was undertaken to identify key features highly correlated with the self-assembly problem. This approach allowed the identification of seven physicochemical and biochemical properties of the amino acids highly associated with the self-assembly of peptides and proteins into amyloid-like fibrils (normalized frequency of β-sheet, normalized frequency of β-sheet from LG, weights for β-sheet at the window position of 1, isoelectric point, atom-based hydrophobic moment, helix termination parameter at position j+1 and ΔG° values for peptides extrapolated in 0 M urea). Moreover, these features enabled the development of a new predictor (available at http://cran.r-project.org/web/packages/appnn/index.html) capable of accurately and reliably predicting the amyloidogenic propensity from the polypeptide sequence alone with a prediction accuracy of 84.9 % against an external validation dataset of sequences with experimental in vitro, evidence of amyloid formation. 相似文献
12.
Amy?P. Guilfoyle Chandrika?N. Deshpande Josep Font Sadurni Miriam-Rose Ash Samuel Tourle Gerhard Schenk Megan?J. Maher Mika Jormakka 《Biophysical journal》2014,107(12):L45-L48
The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release. However, it is unclear whether this conformational change in the G5 loop is a prerequisite for GDP release, or, alternatively, the movement is a consequence of release. To gain additional insight into the sequence of events leading to GDP release, we have created a chimeric protein comprised of Escherichia coli NFeoB and the G5 loop from the human Giα1 protein. The protein chimera retains GTPase activity at a similar level to wild-type NFeoB, and structural analyses of the nucleotide-free and GDP-bound proteins show that the G5 loop adopts conformations analogous to that of the human nucleotide-bound Giα1 protein in both states. Interestingly, isothermal titration calorimetry and stopped-flow kinetic analyses reveal uncoupled nucleotide affinity and release rates, supporting a model where G5 loop movement promotes nucleotide release.The hydrolysis of guanosine triphosphate (GTP) by GTPases, such as the oncoprotein p21 Ras and heterotrimeric Gα proteins, is a critical regulatory activity for cell growth and proliferation (1). Aberrant GTPases are consequently often implicated in tumorigenesis, developmental disorders, and metabolic diseases (2). Critical for the initiation of a GTPase cycle is the release of guanosine diphosphate (GDP), which allows GTP to bind and switch the protein from an inactive to an active conformation. The GTP is subsequently hydrolyzed to GDP and inorganic phosphate, returning the GTPase to an inactive conformation (3).Given that the release of GDP is the fundamental step in the initiation of a GTPase cycle, the detailed mechanism by which it is released has been under intense scrutiny. Studies using double electron-electron resonance, deuterium-exchange, Rosetta energy analysis, and electron paramagnetic resonance, have shown that the mechanism involves conformational changes in the nucleotide-coordinating G5 loop, one of five nucleotide recognition motifs (4, 5, 6, 7, 8, 9, 10, 11). Structural studies of eukaryotic Gα proteins and the intracellular TEES-type GTPase domain of the prokaryotic iron transporter FeoB (NFeoB) have also illustrated distinct conformations of the G5 loop, depending on the nucleotide-bound state (9, 12).Recently, we reported mutational studies of the G5 loop of Escherichia coli NFeoB, which illustrated a correlation between the sequence composition of the loop and the intrinsic GDP release rate (13). However, despite these observations, it is unclear whether the observed conformational changes in the G5 loop are a prerequisite for GDP release, or if the movement is a consequence of GDP release. To address this fundamental question, in this study we have used a combination of protein engineering and biophysical methods.Initially, to assess the relevance of conformational flexibility in the G5 loop, we aimed to create a protein chimera combining sequence and structural characteristics of both fast and slow GDP-releasing GTPases. We thus engineered a protein chimera using E. coli NFeoB as the scaffold (a protein with fast intrinsic GDP release) and substituted the G5 loop with that of a slow GDP-releasing protein (the human Giα1 protein; Gene ID 2770; Fig. 1
A (5)). GTP hydrolysis assays comparing wild-type (wt) NFeoB (wtNFeoB) and the protein chimera (ChiNFeoB) validated the integrity of the GTPase activities of both proteins (kcat = 0.46 and 0.36 min−1, respectively). To further assess the ChiNFeoB protein, we determined its crystal structure at 2.2 Å resolution (see Table S1 in the Supporting Material). The ChiNFeoB structure contains two molecules in the asymmetric unit, with molecule A bound to GDP. They are essentially identical to the nucleotide-bound wtNFeoB structure (root-mean-square deviation of 1.2 Å over 226 Cα atoms; Fig. 2).Open in a separate windowFigure 1Chimera model and structural comparison. (A) Illustration highlighting the chimera sequence change. (Orange) Sequence of the extended G5 loop from Giα1, which replaced the NFeoB sequence (gray). (B–F) Structural comparison of the G5 loop between (B) WT apo (PDB:3HYR) and nucleotide-bound (PDB:3HYT) NFeoB structures. (C) NFeoB nucleotide-bound and Giα1 (PDB:2ZJZ). (D) Nucleotide-bound NFeoB and chimera (Chi_GDP). (E) Nucleotide-bound chimera and Giα1. (F) Nucleotide-free (Chi_apo) and bound chimera protein. (G) Overview of the nucleotide binding site and structural overlay of chimera and Giα1 structures. To see this figure in color, go online.Open in a separate windowFigure 2Superimposition of nucleotide-bound NFeoB and chimera protein, with thermodynamic parameters. To see this figure in color, go online.However, the ChiNFeoB structure, when compared to the wtNFeoB structure, revealed an alteration in the conformation of the G5 loop, showing an extra turn on the N-terminal end of the α6 helix. This is structurally distinct from the wtFeoB protein, but with a conformation similar to that of the Giα1 protein (PDB:2ZJZ; Fig. 1, B–F). As in the crystal structures of wtNFeoB and Giα1, ChiNFeoB residues implicated in coordination of the nucleotide base maintain their positions in the G5 loop relative to GDP. In particular, residues Ala∗150 and Thr∗151 (NFeoB numbering, the asterisk indicates Giα1 chimera residue) are involved in electrostatic interactions with the nucleotide base moiety, analogous to the structures of both wtNFeoB and Giα1 (Fig. 1
G). Serendipitously, the second molecule in the asymmetric unit of ChiNFeoB (molecule B) was present in the nucleotide-free state. The two molecules (GDP-bound and nucleotide-free) are nearly identical (the superposition of molecules A and B yields a root-mean-square deviation of 0.36 Å over 229 Cα atoms), with the G5 loop adopting a nearly indistinguishable conformation compared to that of the GDP-bound molecule A (Fig. 1
F).Importantly, this conformation is independent of the crystallographic packing, inasmuch as the loop is not involved in any crystal contacts. In contrast, the structures of nucleotide-bound and nucleotide-free wtNFeoB illustrated a large conformational change in the G5 loop (Fig. 1
B). Hence, the substitution in the chimera extends the secondary structure of the α6 helix, and as hypothesized, the engineered ChiNFeoB protein has a G5 loop structure that is more conformationally stable than that of wtNFeoB.We subsequently measured the affinity of the ChiNFeoB protein for GDP using isothermal titration calorimetry (ITC). Nonlinear regression was used to attain the thermodynamic parameters (including the GDP binding affinity, Ka; the corresponding dissociation constant (Kd) was calculated from the equation Kd = 1/Ka). Interestingly, these measurements revealed the ChiNFeoB protein to have an almost 10-fold reduced affinity for GDP (82 vs. 9 μM measured for the WT protein; Fig. 2). In contrast, in a recent alanine scanning mutagenesis study of the G5 loop we observed a fivefold increase in affinity for GDP in a Ser150Ala mutant (2 μM) (14). This mutant protein has a coordination environment for the GDP base analogous to that of the ChiNFeoB protein (Fig. 1
A), indicating that it is not the presence of an alanine at position 150 that causes the reduced GDP affinity observed for the chimera protein. Instead, the analysis by ITC and comparison with previous mutagenesis studies indicates that the GDP binding site is less accessible in the ChiNFeoB protein, likely due to the introduction of conformational rigidity that accompanies the extension of secondary structural elements within the loop (Fig. 1
D).To further evaluate the functional characteristics of the chimera protein, we used stopped-flow fluorescence assays to determine the rate of nucleotide dissociation (koff) and association (kon) for the ChiNFeoB protein. The association rate for the GTP analog mant-GMPPNP was determined from the slope of a linear plot of protein concentration versus the observed association constant (kobs). The kon for the chimera was determined to be 3.20 μM−1 min−1 (Supporting Material), the dissociation rate (koff) of GDP for the chimera was determined to be 16.6 s−1 (vs. 144 s−1 for wtNFeoB; Designation mGMPPNP mGDP Protein kona (μM−1 min−1) koffb (min−1) Kdc (μM) kond (μM−1 min−1) koffe (s−1) NFeoB 8.1 ± 0.1 78.6 ± 1.6 9.7 15.9 144.7 ± 2.0 Chimera 3.2 ± 0.1 208.2 ± 1.3 65.1 0.2 16.61 ± 0.50