The pathways of escape of carbon monoxide (CO) from sperm whale myoglobin were investigated by means of a biased form of all‐atoms molecular dynamics (RAMD), whereby a weak, randomly oriented force is applied to the center of mass of CO. The force only persists if the direction taken by CO continues for a given period of time, otherwise a new direction is randomly chosen. A statistically significant number of RAMD runs gave distinct responses according to the level of approximations used for the model. Thus, with rigid bonds to all H‐atoms, several portals for CO egress toward the solvent, besides the main H64 gate, were identified, like in recently published unbiased massive MD, six orders of magnitude acceleration of CO escape in RAMD notwithstanding. In contrast, by removing the approximation of rigid bonds in the model, only one of these extra portals was identified, besides the H64 portal. Sticking to this all‐free‐bonds model, Perutz's early suggestion that the H64 imidazole must rotate ‘out’ toward the solvent in order that CO can cross the H64 gate was directly implemented. RAMD Simulations with this model led to CO egress from the H64 gate only, reconciling theory with experiments. 相似文献
In this work, by applying a non‐deterministic, randomly‐oriented minimal force to the dissociated CO ligand of the MauG‐CO system, the molecular‐dynamics (MD) behavior of this system could be quickly unraveled. It turned out that CO has no marked directional egress from the high‐spin c‐heme iron distal pocket. Rather, CO is able to exploit all interstices created during the protein fluctuations. Nonetheless, no steady route toward the surrounding solvent was ever observed: CO jumped first into other binding pockets before being able to escape the protein. In a few cases, on hitting the surrounding H2O molecules, CO was observed to reverse direction, re‐entering the protein. A contention that conformational inversion of the P107 ring provides a gate to the iron ion is not supported by the present simulations. 相似文献
It is shown here that Fe2+ and O2 ligands are displaced from the ferroxidase center of the C1 four‐helix bundle of E. coli 24‐mer ferritin under molecular dynamics (MD) aided by a randomly oriented external force applied to the ligand. Under these conditions, ligand egress toward the external aqueous medium occurs preferentially from the same four‐helix bundle, in the case of O2, or other bundle, in the case of Fe2. Viewing ligand egress from the protein as the microscopic reverse of ligand influx into the protein under unbiased MD, these findings challenge current views that preferential gates for recruitment of Fe2+ are 3‐fold channels with human ferritin, or the short path from the ferroxidase center to H93 with bacterial ferritins. 相似文献
This work deals with a trimeric bacterial protein, RhCC, which, although belonging to the tautomerase superfamily, shows oxygenase activity. A model of the complex from RhCC and substrate 4‐hydroxyphenylenolpyruvate (4HPP), fitting the observation of extra electron densities from X‐ray diffraction of the crystal, could be built by autodocking. When subjected to molecular dynamics (MD) aided by an external random force applied to a O2 molecule placed above 4HPP, this model evolved with O2 egressing toward the bulk solvent from two nearly opposite gates. These were located between the nearly parallel helices 75 – 91 and 15 – 33 of either chain C (gate SE) or chain B (gate FL). Alternatively, with four O2 molecules in the bulk solvent, unbiased MD led to O2 entering the protein from gate SE and getting to 4HPP, while forming a stabilizing salt bridge between the 4HPP carboxylate and P1.C +NH2, thus providing scientific ground for a refined model of the complex. 相似文献
H‐NOX (Heme Nitric Oxide/Oxygen) domain has widespread occurrence, either standalone or associated with functional proteins, sending signals for functions that span from modulating vasodilation and neurotransmission with humans to competition and symbiosis with bacteria. Understanding how H‐NOX works, and possibly intervening on degeneration for health purposes, needs first clarifying how diatomic gases are relocated through this protein in relation to the deeply buried heme. To this end, a biased form of molecular dynamics, i.e., Random Accelaration Molecular Dynamics (RAMD), is used by applying a randomly oriented tiny force to heme‐dissociated CO of Nostoc sp. H‐NOX, while changing randomly the direction of the force, if CO travels less than specified for the evaluated block. The result is that a large area of the protein, comprising amino acids from serine 44 to leucine 67 along two adjacent helices, offers a broad portal to CO from the surrounding medium to the deeply buried heme. Most traffic is concentrated through a channel lined by tyrosine 49, valine 52, and leucine 67. This modifies the picture drawn from mapping Xe cavities on pressurizing Nostoc sp. H‐NOX with Xe gas. What is the main pathway with Xe‐cavity mapping becomes a minor pathway with RAMD, and vice versa. The reason is that the fluctuating protein under MD creates clefts for CO slipping through, as it is expected to occur in nature. 相似文献
Heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), a vascular‐derived trophic factor, belongs to the epidermal growth factor (EGF) family of neuroprotective, hypoxia‐inducible proteins released by astrocytes in CNS injuries. It was suggested that HB–EGF can replace fetal calf serum (FCS) in astrocyte cultures. We previously demonstrated that in contrast to standard 2D cell culture systems, Bioactive3D culture system, when used with FCS, minimizes the baseline activation of astrocytes and preserves their complex morphology. Here, we show that HB‐EGF induced EGF receptor (EGFR) activation by Y1068 phosphorylation, Mapk/Erk pathway activation, and led to an increase in cell proliferation, more prominent in Bioactive3D than in 2D cultures. HB‐EGF changed morphology of 2D and Bioactive3D cultured astrocytes toward a radial glia‐like phenotype and induced the expression of intermediate filament and progenitor cell marker protein nestin. Glial fibrillary acidic protein (GFAP) and vimentin protein expression was unaffected. RT‐qPCR analysis demonstrated that HB‐EGF affected the expression of Notch signaling pathway genes, implying a role for the Notch signaling in HB‐EGF‐mediated astrocyte response. HB‐EGF can be used as a FCS replacement for astrocyte expansion and in vitro experimentation both in 2D and Bioactive3D culture systems; however, caution should be exercised since it appears to induce partial de‐differentiation of astrocytes.
We investigated the relationship of polymorphisms in the cholecystokinin 1 receptor [CCK1R; G to T (n‐128), A to G (n‐81)] and the β3‐adrenergic receptor (β3‐AR; Trp64Arg) with midlife weight gain. The participants were 1012 Japanese men and women (40 to 59 years of age). Their weight at 18 years old was obtained from a questionnaire. Weight change was defined as the current weight minus the weight at 18 years old. Subjects were grouped into four categories by these genotypes: W/W = noncarriers, W/H = Arg64 carriers of the β3‐AR, H/W = T (n‐128) or G (n‐81) carriers of the CCK1R, H/H = T (n‐128) or G (n‐81) and Arg64 carriers. In men, the interaction between the CCK1R and β3‐AR polymorphisms was significant (two‐way ANOVA, p < 0.05), but neither the CCK1R nor the β3‐AR was individually associated with weight gain. The H/H group showed a higher possibility of weight gain of 10 kg or more compared with the W/W group in men. The odds ratio for weight gain (≥10 kg) of H/H was 2.54 (95% confidence interval: 1.50 to 4.30) compared with W/W. In women, neither main effect nor interaction was significant. These results suggest that the combination of CCK1R and the β3‐AR polymorphisms is a contributing factor for midlife weight gain in men. 相似文献
Using degenerate primers based on published 2-microglobulin sequences we were able to obtain an expected 111 base pairs (bp) polymerase chain reaction (PCR) fragment from tilapia genomic DNA. The sequence of this fragment showed a high degree of similarity to mouse 2-microglobulin at the protein level. We used these primers in an anchored PCR to obtain a 213 bp PCR fragment from a carp cDNA library. This was then used to clone a full-length 2-microglobulin cDNA from carp. The carp sequence showed the highest similarity to rabbit 2-microglobulin. Both sequences showed strong similarities to all previously published vertebrate 2-microglobulin sequences. The predicted protein secondary structure of both the carp and tilapia clones was almost identical to the corresponding regions of previously known vertebrate 2-microglobulin protein sequences. When either the carp or tilapia probes were used against corresponding northern blots, they hybridized to a message of approximately 800–1000 bases long, which corresponds to the previously published lengths of 2-microglobulin mRNAs. Southern blotting indicated that 2-microglobulin was encoded by a single copy gene in both cases. Phylogenetic analysis indicated that the sequences were related to the 2-microglobulins of higher vertebrates but grouped together in an ancestral position.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L05536 (carp), L05537 (tilapia). 相似文献
It is reported here on random acceleration molecular dynamics (RAMD) simulations with the 2GF3 bacterial monomeric sarcosine oxidase (MSOX), O2, and furoic acid in place of sarcosine, solvated by TIP3 H2O in a periodic box. An external tiny force, acting randomly on O2, accelerated its relocation, from the center of activation between residue K265 and the si face of the flavin ring of the flavin adenine dinucleotide cofactor, to the surrounding solvent. Only three of the four O2 gates previously described for this system along a composite method technique were identified, while two more major O2 gates were found. The RAMD simulations also revealed that the same gate can be reached by O2 along different pathways, often involving traps for O2. Both the residence time of O2 in the traps, and the total trajectory time for O2 getting to the solvent, could be evaluated. The new quick pathways discovered here suggest that O2 exploits all nearby interstices created by the thermal fluctuations of the protein, not having necessarily to look for the permanent large channel used for uptake of the FADH cofactor. To this regard, MSOX resembles closely KijD3 N‐oxygenase. These observations solicit experimental substantiation, in a long term aim at discovering whether gates and pathways for the small gaseous ligands inside the proteins are under Darwinian functional evolution or merely stochastic control operates. 相似文献
The effect of the Y108V mutation of human glutathione S‐transferase P1‐1 (hGST P1‐1) on the binding of the diuretic drug ethacrynic acid (EA) and its glutathione conjugate (EASG) was investigated by calorimetric, spectrofluorimetric, and crystallographic studies. The mutation Tyr 108 → Val resulted in a 3D‐structure very similar to the wild type (wt) enzyme, where both the hydrophobic ligand binding site (H‐site) and glutathione binding site (G‐site) are unchanged except for the mutation itself. However, due to a slight increase in the hydrophobicity of the H‐site, as a consequence of the mutation, an increase in the entropy was observed. The Y108V mutation does not affect the affinity of EASG for the enzyme, which has a higher affinity (Kd ~ 0.5 μM) when compared with those of the parent compounds, K ~ 13 μM, K ~ 25 μM. The EA moiety of the conjugate binds in the H‐site of Y108V mutant in a fashion completely different to those observed in the crystal structures of the EA or EASG wt complex structures. We further demonstrate that the ΔCp values of binding can also be correlated with the potential stacking interactions between ligand and residues located in the binding sites as predicted from crystal structures. Moreover, the mutation does not significantly affect the global stability of the enzyme. Our results demonstrate that calorimetric measurements maybe useful in determining the preference of binding (the binding mode) for a drug to a specific site of the enzyme, even in the absence of structural information. 相似文献
Summary The energy requirements of Adélie penguin (Pygoscelis adeliae) chicks were analysed with respect to body mass (W, 0.145–3.35 kg, n=36) and various forms of activity (lying, standing, minor activity, locomotion, walking on a treadmill). Direct respirometry was used to measure O2 consumption (
) and CO2 production. Heart rate (HR, bpm) was recorded from the ECG obtained by both externally attached electrodes and implantable HR-transmitters. The parameters measured were not affected by hand-rearing of the chicks or by implanting transmitters. HR measured in the laboratory and in the field were comparable. Oxygen uptake ranged from
in lying chicks to
at maximal activity, RQ=0.76. Metabolic rate in small wild chicks (0.14–0.38 kg) was not affected by time of day, nor was their feeding frequency in the colony (Dec 20–21). Regressions of HR on
were highly significant (p< 0.0001) in transmitter implanted chicks (n=4), and two relationships are proposed for the pooled data, one for minor activities (
), and one for walking (
). Oxygen consumption, mass of the chick (2–3 kg), and duration of walking (T, s) were related as
, whereas mass-specific O2 consumption was related to walking speed (S, m·s-1) as
.Abbreviations
bpm
beats per minute
-
D
distance walked (m)
-
ECG
electrocardiogram
-
HR
heart rate (bpm)
-
ns
number of steps
-
RQ
respiratory quotient
-
S
walking speed (m·s-1)
-
T
time walked (s)
-
W
body mass (kg) 相似文献
Anxiety disorders are associated with a high social burden worldwide. Recently, increasing evidence suggests that nuclear factor kappa B (NF‐κB) has significant implications for psychiatric diseases, including anxiety and depressive disorders. However, the molecular mechanisms underlying the role of NF‐κB in stress‐induced anxiety behaviors are poorly understood. In this study, we show that chronic mild stress (CMS) and glucocorticoids dramatically increased the expression of NF‐κB subunits p50 and p65, phosphorylation and acetylation of p65, and the level of nuclear p65 in vivo and in vitro , implicating activation of NF‐κB signaling in chronic stress‐induced pathological processes. Using the novelty‐suppressed feeding (NSF) and elevated‐plus maze (EPM) tests, we found that treatment with pyrrolidine dithiocarbamate (PDTC; intra‐hippocampal infusion), an inhibitor of NF‐κB, rescued the CMS‐ or glucocorticoid‐induced anxiogenic behaviors in mice. Microinjection of PDTC into the hippocampus reversed CMS‐induced up‐regulation of neuronal nitric oxide synthase (nNOS), carboxy‐terminal PDZ ligand of nNOS (CAPON), and dexamethasone‐induced ras protein 1 (Dexras1) and dendritic spine loss of dentate gyrus (DG) granule cells. Moreover, over‐expression of CAPON by infusing LV‐CAPON‐L‐GFP into the hippocampus induced nNOS‐Dexras1 interaction and anxiety‐like behaviors, and inhibition of NF‐κB by PDTC reduced the LV‐CAPON‐L‐GFP‐induced increases in nNOS‐Dexras1 complex and anxiogenic‐like effects in mice. These findings indicate that hippocampal NF‐κB mediates anxiogenic behaviors, probably via regulating the association of nNOS‐CAPON‐Dexras1, and uncover a novel approach to the treatment of anxiety disorders.
The recently defined
versus
straight-line plots for L = pyridine-type (PyN) and
ortho-aminopyridine-type (oPyN) ligands now allow the evaluation in a quantitative manner of the stability of the 1:1 complexes formed between cytidine (Cyd) and Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+
(M2+); the corresponding stability constants,
, including the acidity constant,
, for the deprotonation of the (N3)H+ site had been determined previously under exactly the same conditions as the mentioned plots. Since the stabilities of the M(PyN)2+ and M(oPyN)2+ complexes of Ca2+ and Mg2+ are practically identical, it is concluded that complex formation occurs in an outer-sphere manner, and this is in accord with the fact that in the pKa range 3–7 metal ion binding is independent of
or
. Ca(Cyd)2+
and Mg(Cyd)2+ are more stable than the corresponding (outer-sphere) M(PyN)2+ complexes and this means that the C2 carbonyl group of Cyd must participate, next to N3 which is most likely outer-sphere, in metal ion binding, leading thus to chelates; these have formation degrees of about 50% and 35%, respectively. Co(Cyd)2+ and Ni(Cyd)2+
show no increased stability based on the
hence, the (C2)O group does not participate in metal ion binding, but the inner-sphere coordination to N3 is strongly inhibited by the (C4)NH2 group. In the M(Cyd)2+ complexes of Mn2+, Cu2+, Zn2+ and Cd2+, this inhibiting effect on M2+ binding at N3 is partially compensated by participation of the (C2)O group in complex formation and the corresponding chelates have formation degrees between about 30% (Zn2+) and 83% (Cu2+). The different structures of the mentioned chelates are discussed in relation to available crystal structure analyses. (1) There is evidence (crystal structure studies: Cu2+, Zn2+, Cd2+) that four-membered rings form, i.e. there is a strong M2+ bond to N3 and a weak one to (C2)O. (2) By hydrogen bond formation to (C2)O of a metal ion-bound water molecule, six-membered rings, so-called semichelates, may form. (3) For Ca2+ and Mg2+, and possibly Mn2+, and their Cyd complexes, six-membered chelates are also likely with (C2)O being inner-sphere (crystal structure) and N3 outer-sphere. (4) Finally, for these metal ions also complexes with a sole outer-sphere interaction may occur. All these types of chelates are expected to be in equilibrium with each other in solution, but, depending on the metal ion, either the one or the other form will dominate. Clearly, the cytidine residue is an ambivalent binding site which adjusts well to the requirements of the metal ion to be bound and this observation is of relevance for single-stranded nucleic acids and their interactions with metal ions. In addition, the anti–syn energy barrier has been estimated as being in the order of 6–7.5 kJ/mol for cytidine derivatives in aqueous solution at 25 °C.Abbreviations ADP3– adenosine 5-diphosphate - AMP2– adenosine 5-monophosphate - ATP4–
adenosine 5-triphosphate - CDP3–
cytidine 5-diphosphate - cl
closed - CMP2– cytidine 5-monophosphate - 3-CMP2– cytidine 3-monophosphate - CTP4–
cytidine 5-triphosphate - Cyd
cytidine - DNA deoxyribonucleic acid - I ionic strength - Ka acidity constant - KI intramolecular equilibrium constant - L general ligand - M2+ general divalent metal ion - NTP4–
nucleoside 5-triphosphate - op
open - oPyN
ortho-aminopyridine-type ligand - PyN pyridine-type ligand - t-RNA
transfer ribonucleic acid - Tu tubercidin (7-deazaadenosine)In honor of Professor Liang-Nian Ji on the occasion of his 70th birthday in friendship and with best wishes. 相似文献
Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch
clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity
of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied
here included the well-known lipophilic anions dipicrylamine (DPA−), tetraphenylborate (TPB−) and [W2(CO)10(S2CH)]−, the putative lipophilic anion
and three new heterocyclic W(CO)5 derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped
cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum
capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption
depth of the large organic anions DPA−, TPB− and increased and that of W(CO)5 derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation
of cells treated with DPA− and W(CO)5 derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation
of mobile charges. In contrast, in the presence of TPB− and no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the
presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic
anions. 相似文献