共查询到20条相似文献,搜索用时 26 毫秒
1.
Balakrishnan?S. Moorthy Hamed?Tabatabaei Ghomi Markus?A. Lill Elizabeth?M. Topp 《Biophysical journal》2015,108(4):937-948
A mechanistic understanding of the intermolecular interactions and structural changes during fibrillation is crucial for the design of safe and efficacious glucagon formulations. Amide hydrogen/deuterium exchange with mass spectrometric analysis was used to identify the interactions and amino acids involved in the initial stages of glucagon fibril formation at acidic pH. Kinetic measurements from intrinsic and thioflavin T fluorescence showed sigmoidal behavior. Secondary structural measurement of fibrillating glucagon using far-UV circular dichroism spectroscopy showed changes in structure from random coil → α-helix → β-sheet, with increase in α-helix content during the lag phase followed by increase in β-sheet content during the growth phase. Hydrogen/deuterium exchange with mass spectrometric analysis of fibrillating glucagon suggested that C-terminal residues 22–29 are involved in interactions during the lag phase, during which N-terminal residues 1–6 showed no changes. Molecular dynamics simulations of glucagon fragments showed C-terminal to C-terminal interactions with greater α-helix content for the 20–29 fragment, with hydrophobic and aromatic residues (Phe-22, Trp-25, Val-23, and Met-27) predominantly involved. Overall, the study shows that glucagon interactions during the early phase of fibrillation are mediated through C-terminal residues, which facilitate the formation of α-helix-rich oligomers, which further undergo structural rearrangement and elongation to form β-sheet-rich mature fibrils. 相似文献
2.
Using isobaric-isothermal replica-exchange molecular dynamics and the all-atom explicit-solvent model, we studied the equilibrium binding of Aβ monomers to a zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer coincubated with calcium ions. Using our previous replica-exchange molecular dynamics calcium-free simulations as a control, we reached three conclusions. First, calcium ions change the tertiary structure of the bound Aβ monomer by destabilizing several long-range intrapeptide interactions, particularly the salt bridge Asp23-Lys28. Second, calcium strengthens Aβ peptide binding to the DMPC bilayer by enhancing electrostatic interactions between charged amino acids and lipid polar headgroups. As a result, Aβ monomer penetrates deeper into the bilayer, making disorder in proximal lipids and bilayer thinning more pronounced. Third, because calcium ions demonstrate strong affinity to negatively charged amino acids, a considerable influx of calcium into the area proximal to the bound Aβ monomer is observed. Consequently, the localizations of negatively charged amino acids and calcium ions in the Aβ binding footprint overlap. Based on our data, we propose a mechanism by which calcium ions strengthen Aβ-bilayer interactions. This mechanism involves two factors: 1) calcium ions make the DMPC bilayer partially cationic and thus attractive to the anionic Aβ peptide; and 2) destabilization of the Asp23-Lys28 salt bridge makes Lys28 available for interactions with the bilayer. Finally, we conclude that a single Aβ monomer does not promote permeation of calcium ions through the zwitterionic bilayer. 相似文献
3.
The torque of bacterial flagellar motors is generated by interactions between the rotor and the stator and is coupled to the influx of H+ or Na+ through the stator. A chimeric protein, PotB, in which the N-terminal region of Vibrio alginolyticus PomB was fused to the C-terminal region of Escherichia coli MotB, can function with PomA as a Na+-driven stator in E. coli. Here, we constructed a deletion variant of PotB (with a deletion of residues 41 to 91 [Δ41–91], called PotBΔL), which lacks the periplasmic linker region including the segment that works as a “plug” to inhibit premature ion influx. This variant did not confer motile ability, but we isolated a Na+-driven, spontaneous suppressor mutant, which has a point mutation (R109P) in the MotB/PomB-specific α-helix that connects the transmembrane and peptidoglycan binding domains of PotBΔL in the region of MotB. Overproduction of the PomA/PotBΔL(R109P) stator inhibited the growth of E. coli cells, suggesting that this stator has high Na+-conducting activity. Mutational analyses of Arg109 and nearby residues suggest that the structural alteration in this α-helix optimizes PotBΔL conformation and restores the proper arrangement of transmembrane helices to form a functional channel pore. We speculate that this α-helix plays a key role in assembly-coupled stator activation. 相似文献
4.
Jennifer C. Mortensen Jovan Damjanovic Jiayuan Miao Tiffani Hui YuShan Lin 《Protein science : a publication of the Protein Society》2022,31(12)
Backbone‐dependent rotamer libraries are commonly used to assign the side chain dihedral angles of amino acids when modeling protein structures. Most rotamer libraries are created by curating protein crystal structure data and using various methods to extrapolate the existing data to cover all possible backbone conformations. However, these rotamer libraries may not be suitable for modeling the structures of cyclic peptides and other constrained peptides because these molecules frequently sample backbone conformations rarely seen in the crystal structures of linear proteins. To provide backbone‐dependent side chain information beyond the α‐helix, β‐sheet, and PPII regions, we used explicit‐solvent metadynamics simulations of model dipeptides to create a new rotamer library that has high coverage in the (ϕ, ψ) space. Furthermore, this approach can be applied to build high‐coverage rotamer libraries for noncanonical amino acids. The resulting Metadynamics of Dipeptides for Rotamer Distribution (MEDFORD) rotamer library predicts the side chain conformations of high‐resolution protein crystal structures with similar accuracy (~80%) to a state‐of‐the‐art rotamer library. Our ability to test the accuracy of MEDFORD at predicting the side chain dihedral angles of amino acids in noncanonical backbone conformation is restricted by the limited structural data available for cyclic peptides. For the cyclic peptide data that are currently available, MEDFORD and the state‐of‐the‐art rotamer library perform comparably. However, the two rotamer libraries indeed make different rotamer predictions in noncanonical (ϕ, ψ) regions. For noncanonical amino acids, the MEDFORD rotamer library predicts the χ 1 values with approximately 75% accuracy. 相似文献
5.
Sougata Ghosh Piyush More Abhishek Derle Ajay B. Patil Pramod Markad Adersh Asok Navanath Kumbhar Mahemud L. Shaikh Boppana Ramanamurthy Vaishali S. Shinde Dilip D. Dhavale Balu A. Chopade 《PloS one》2014,9(9)
Diabetes mellitus is a multifactorial metabolic disease characterized by post-prandial hyperglycemia (PPHG). α-amylase and α-glucosidase inhibitors aim to explore novel therapeutic agents. Herein we report the promises of Dioscorea bulbifera and its bioactive principle, diosgenin as novel α-amylase and α-glucosidase inhibitor. Among petroleum ether, ethyl acetate, methanol and 70% ethanol (v/v) extracts of bulbs of D. bulbifera, ethyl acetate extract showed highest inhibition upto 72.06 ± 0.51% and 82.64 ± 2.32% against α-amylase and α-glucosidase respectively. GC-TOF-MS analysis of ethyl acetate extract indicated presence of high diosgenin content. Diosgenin was isolated and identified by FTIR, 1H NMR and 13C NMR and confirmed by HPLC which showed an α-amylase and α-glucosidase inhibition upto 70.94 ± 1.24% and 81.71 ± 3.39%, respectively. Kinetic studies confirmed the uncompetitive mode of binding of diosgenin to α-amylase indicated by lowering of both Km and Vm. Interaction studies revealed the quenching of intrinsic fluorescence of α-amylase in presence of diosgenin. Similarly, circular dichroism spectrometry showed diminished negative humped peaks at 208 nm and 222 nm. Molecular docking indicated hydrogen bonding between carboxyl group of Asp300, while hydrophobic interactions between Tyr62, Trp58, Trp59, Val163, His305 and Gln63 residues of α-amylase. Diosgenin interacted with two catalytic residues (Asp352 and Glu411) from α-glucosidase. This is the first report of its kind that provides an intense scientific rationale for use of diosgenin as novel drug candidate for type II diabetes mellitus. 相似文献
6.
Gunhild M. Mueller Ahmad B. Maarouf Carol L. Kinlough Nan Sheng Ossama B. Kashlan Sora Okumura Sarah Luthy Thomas R. Kleyman Rebecca P. Hughey 《The Journal of biological chemistry》2010,285(40):30453-30462
The epithelial Na+ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na+ self-inhibition, and reduced single channel Po when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane. 相似文献
7.
pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides
下载免费PDF全文
![点击此处可从《Protein science : a publication of the Protein Society》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Toshiaki Takei Kouhei Tsumoto Atsuhito Okonogi Akiko Kimura Shuichi Kojima Kazumori Yazaki Tsunetomo Takei Takuya Ueda Kin-ichiro Miura 《Protein science : a publication of the Protein Society》2015,24(5):883-894
We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation. 相似文献
8.
Wei Yan Qing Ye Min Tan Xi Chen Gilbert Eriani En-Duo Wang 《The Journal of biological chemistry》2015,290(19):12256-12267
A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNALeu, which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNALeu D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. 相似文献
9.
Nebojsa Murisic Vincent Hakim Ioannis?G. Kevrekidis Stanislav?Y. Shvartsman Basile Audoly 《Biophysical journal》2015,108(1):154-162
Metamorphic proteins, including proteins with high levels of sequence identity but different folds, are exceptions to the long-standing rule-of-thumb that proteins with as little as 30% sequence identity adopt the same fold. Which topologies can be bridged by these highly identical sequences remains an open question. Here we bridge two 3-α-helix bundle proteins with two radically different folds. Using a straightforward approach, we engineered the sequences of one subdomain within maltose binding protein (MBP, α/β/α-sandwich) and another within outer surface protein A (OspA, β-sheet) to have high sequence identity (80 and 77%, respectively) with engineered variants of protein G (GA, 3-α-helix bundle). Circular dichroism and nuclear magnetic resonance spectra of all engineered variants demonstrate that they maintain their native conformations despite substantial sequence modification. Furthermore, the MBP variant (80% identical to GA) remained active. Thermodynamic analysis of numerous GA and MBP variants suggests that the key to our approach involved stabilizing the modified MBP and OspA subdomains via external interactions with neighboring substructures, indicating that subdomain interactions can stabilize alternative folds over a broad range of sequence variation. These findings suggest that it is possible to bridge one fold with many other topologies, which has implications for protein folding, evolution, and misfolding diseases. 相似文献
10.
DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein α. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in α (αcr) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on λ immunity in E.coli for in vivo detection of protein–protein interactions, we found that: (i) α protein interacts with Cnr, whereas αcr proteins do not; (ii) both α–α and αcr–αcr interactions occur and the interaction domain is located within the C-terminal of α; (iii) Cnr–Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both α–α and αcr–αcr dimerization. Our data suggest that Cnr and α interact in at least two ways, which may have different functional roles in P4 replication control. 相似文献
11.
12.
The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m−1·s−1) to produce a relatively stable complex (t½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH. 相似文献
13.
Croke RL Sallum CO Watson E Watt ED Alexandrescu AT 《Protein science : a publication of the Protein Society》2008,17(8):1434-1445
Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in 1H-15N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils. 相似文献
14.
Mackenzie J. Thompson Jaimee A. Domville Claire H. Edrington Angelica Venes Patrick M. Gigure John E. Baenziger 《The Journal of biological chemistry》2022,298(7)
The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4–M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4–M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function. 相似文献
15.
Ca2+ Current and Charge Movements in Skeletal Myotubes Promoted by the β-Subunit of the Dihydropyridine Receptor in the Absence of Ryanodine Receptor Type 1
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Chris A. Ahern David. C. Sheridan Weijun Cheng Lindsay Mortenson Priya Nataraj Paul Allen Michel De Waard Roberto Coronado 《Biophysical journal》2003,84(2):942-959
The β-subunit of the dihydropyridine receptor (DHPR) enhances the Ca2+ channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the β-subunit in the expression of L-type Ca2+ current and charge movements in RyR1 knock-out (KO), β1 KO, and double β1/RyR1 KO myotubes generated by mating heterozygous β1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various β-isoforms and then whole-cell voltage-clamped for measurements of Ca2+ and gating currents. Overexpression of the endogenous skeletal β1a isoform resulted in a low-density Ca2+ current either in RyR1 KO (36 ± 9 pS/pF) or in β1/RyR1 KO (34 ± 7 pS/pF) myotubes. However, the heterologous β2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca2+ current that was entirely wild-type in density in RyR1 KO (195 ± 16 pS/pF) and was significantly enhanced in double β1/RyR1 KO (115 ± 18 pS/pF) myotubes. Other variants tested from the four β gene families (β1a, β1b, β1c, β3, and β4) were unable to enhance Ca2+ current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in β2a-expressing β1a/RyR1 KO myotubes were significantly lower than in β1a-expressing β1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, β2a had a preferential ability to recover Ca2+ current, whereas β1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (β2a C3,4S) eliminated the RyR1-independent Ca2+ current expression. Furthermore, Ca2+ current enhancement was observed with a β2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the β2a variant to the myotube surface activated the DHPR Ca2+ current and bypassed the requirement for RyR1. The data suggest that the Ca2+ current expressed by the native skeletal DHPR complex has an inherently low density due to inhibitory interactions within the DHPR and that the β1a-subunit is critically involved in process. 相似文献
16.
FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis. 相似文献
17.
Shailaja Kunda Yue Yuan Rashna D. Balsara Jaroslav Zajicek Francis J. Castellino 《The Journal of biological chemistry》2015,290(29):18156-18172
Conantokins are ∼20-amino acid peptides present in predatory marine snail venoms that function as allosteric antagonists of ion channels of the N-methyl-d-aspartate receptor (NMDAR). These peptides possess a high percentage of post-/co-translationally modified amino acids, particularly γ-carboxyglutamate (Gla). Appropriately spaced Gla residues allow binding of functional divalent cations, which induces end-to-end α-helices in many conantokins. A smaller number of these peptides additionally contain 4-hydroxyproline (Hyp). Hyp should prevent adoption of the metal ion-induced full α-helix, with unknown functional consequences. To address this disparity, as well as the role of Hyp in conantokins, we have solved the high resolution three-dimensional solution structure of a Gla/Hyp-containing 18-residue conantokin, conRl-B, by high field NMR spectroscopy. We show that Hyp10 disrupts only a small region of the α-helix of the Mn2+·peptide complex, which displays cation-induced α-helices on each terminus of the peptide. The function of conRl-B was examined by measuring its inhibition of NMDA/Gly-mediated current through NMDAR ion channels in mouse cortical neurons. The conRl-B displays high inhibitory selectivity for subclasses of NMDARs that contain the functionally important GluN2B subunit. Replacement of Hyp10 with N8Q results in a Mg2+-complexed end-to-end α-helix, accompanied by attenuation of NMDAR inhibitory activity. However, replacement of Hyp10 with Pro10 allowed the resulting peptide to retain its inhibitory property but diminished its GluN2B specificity. Thus, these modified amino acids, in specific peptide backbones, play critical roles in their subunit-selective inhibition of NMDAR ion channels, a finding that can be employed to design NMDAR antagonists that function at ion channels of distinct NMDAR subclasses. 相似文献
18.
In the present study, glutaryl-7-amino cephalosporanic acid acylase from Pseudomonas sp. strain 130 (CA130) was mutated to improve its enzymatic activity and stability. Based on the crystal structure of CA130, two series of amino acid residues, one from those directly involved in catalytic function and another from those putatively involved in surface charge, were selected as targets for site-directed mutagenesis. In the first series of experiments, several key residues in the substrate-binding pocket were substituted, and the genes were expressed in Escherichia coli for activity screening. Two of the mutants constructed, Y151αF and Q50βN, showed two- to threefold-increased catalytic efficiency (kcat/Km) compared to wild-type CA130. Their Km values were decreased by ca. 50%, and the kcat values increased to 14.4 and 16.9 s−1, respectively. The ability of these mutants to hydrolyze adipoyl 6-amino penicillinic acid was also improved. In the second series of mutagenesis, several mutants with enhanced stabilities were identified. Among them, R121βA and K198βA had a 30 to 58% longer half-life than wild-type CA130, and K198βA and D286βA showed an alkaline shift of optimal pH by about 1.0 to 2.0 pH units. To construct an engineered enzyme with the properties of both increased activity and stability, the double mutant Q50βN/K198βA was expressed. This enzyme was purified and immobilized for catalytic analysis. The immobilized mutant enzyme showed a 34.2% increase in specific activity compared to the immobilized wild-type CA130. 相似文献
19.
Transient responses of a space-clamped squid axon membrane to step changes of voltage or current are often approximated by exponential functions of time, corresponding to a series resistance and a membrane capacity of 1.0 μF/cm2. Curtis and Cole (1938, J. Gen. Physiol. 21:757) found, however, that the membrane had a constant phase angle impedance z = z1(jωτ)-α, with a mean α = 0.85. (α = 1.0 for an ideal capacitor; α < 1.0 may represent dielectric loss.) This result is supported by more recently published experimental data. For comparison with experiments, we have computed functions expressing voltage and current transients with constant phase angle capacitance, a parallel leakage conductance, and a series resistance, at nine values of α from 0.5 to 1.0. A series in powers of tα provided a good approximation for short times; one in powers of t-α, for long times; for intermediate times, a rational approximation matching both series for a finite number of terms was used. These computations may help in determining experimental series resistances and parallel leakage conductances from membrane voltage or current clamp data. 相似文献
20.
Long-Range Correlations of Global Sea Surface Temperature 总被引:1,自引:0,他引:1
Scaling behaviors of the global monthly sea surface temperature (SST) derived from 1870–2009 average monthly data sets of Hadley Centre Sea Ice and SST (HadISST) are investigated employing detrended fluctuation analysis (DFA). The global SST fluctuations are found to be strong positively long-range correlated at all pertinent time-intervals. The value of scaling exponent is larger in the tropics than those in the intermediate latitudes of the northern and southern hemispheres. DFA leads to the scaling exponent α = 0.87 over the globe (60°S~60°N), northern hemisphere (0°N~60°N), and southern hemisphere (0°S~60°S), α = 0.84 over the intermediate latitude of southern hemisphere (30°S~60°S), α = 0.81 over the intermediate latitude of northern hemisphere (30°N~60°N) and α = 0.90 over the tropics 30°S~30°N [fluctuation F(s) ~ sα], which the fluctuations of monthly SST anomaly display long-term correlated behaviors. Furthermore, the larger the standard deviation is, the smaller long-range correlations (LRCs) of SST in the corresponding regions, especially in three distinct upwelling areas. After the standard deviation is taken into account, an index χ = α * σ is introduced to obtain the spatial distributions of χ. There exists an obvious change of global SST in central east and northern Pacific and the northwest Atlantic. This may be as a clue on predictability of climate and ocean variabilities. 相似文献