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11.
H. Raghuraman Shahidul M. Islam Soumi Mukherjee Benoit Roux Eduardo Perozo 《Proceedings of the National Academy of Sciences of the United States of America》2014,111(5):1831-1836
In K+ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K+ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K+ channels.The functional behavior of K+ channels is defined by a series of structural rearrangements associated with the processes of activation and inactivation gating (1–6). In response to a prolonged stimulus and in the absence of an N-terminal inactivating particle, most K+ channels become nonconductive through a process known as C-type inactivation (7). This C-type inactivation is crucial in controlling the firing patterns in excitable cells and is fundamental in determining the length and frequency of the cardiac action potential (8). C-type inactivation is inhibited by high extracellular K+ (9, 10), and the blocker tetraethylammonium (TEA) (11) can also be slowed down in the presence of permeant ions with a long residence time in the selectivity filter (Rb+, Cs+, and NH4+) (10).The prokaryotic pH-gated K+ channel KcsA shares most of the mechanistic properties of C-type inactivation in voltage-dependent K+ channels (5, 6, 12–16). Recent crystal structures of open/inactivated KcsA reveal that there is a remarkable correlation between the degree of opening at the activation gate and the conformation and ion occupancy of the selectivity filter (5). In KcsA, the selectivity filter is stabilized by a hydrogen bond network, with key interactions between residues Glu71, Asp80, and Trp67 and a bound water molecule (17). Disrupting this hydrogen bond network favors the conductive conformation of the selectivity filter (12, 13, 15).Early electrophysiological experiments have suggested that the outer vestibule (around T449 residue in Shaker and Y82 residue in KcsA) undergoes significant conformational rearrangement during C-type inactivation gating (16, 18, 19). However, comparison of the WT KcsA crystal structure, where the filter is in its conductive conformation, with either the structure obtained with low K+ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5) does not show major conformational changes in the outer vestibule that would explain these results (Fig. 1A). We have suggested that this apparent discrepancy can be understood if we take into consideration the potential differences in the dynamic behavior of the outer vestibule changes as the K+ channel undergoes its gating cycle (16).Open in a separate windowFig. 1.Comparison of outer vestibule conformation in KcsA structures with conductive and collapsed/inactivated filters. (A) High-K+ KcsA structure [Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K+ KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outer vestibule residues are depicted as red spheres, and relevant residues are labeled. (B) Schematic representation of typical macroscopic currents elicited by pH-jump experiments in WT (inactivating) and E71A (noninactivating) KcsA channels at a depolarizing membrane potential is shown. Conditions that stabilize the closed, open/inactivated, and open/conductive conformations at the steady state are indicated with a black circle. (C) Effect of opening the lower gate on the mobility of spin-labeled outer vestibule residues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidyl glycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and noninactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) and open (pH 4, black) states of KcsA, as determined by continuous wave (CW) EPR. The spectra shown are amplitude-normalized. Details are provided in SI Materials and Methods.We have probed the gating-induced structural dynamics at the outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combination with a recently developed computational method, restrained ensemble (RE) simulations. RE simulation was used to constrain the outer vestibule using experimentally derived distance histograms in different functional states (closed, open/inactivated, and open/conductive) and to monitor the extent of backbone conformational changes during gating. To this end, we took advantage of our ability to stabilize both the open/conductive (E71A mutant) and the open/inactivated (WT) conformations of KcsA upon opening the activation gate under steady-state conditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive conformation is highly dynamic. In addition, the red edge excitation shift (REES) points to a change in hydration dynamics between conductive and nonconductive outer vestibule conformations, suggesting a role of restricted water molecules in C-type inactivation gating. We suggest that, on average, the backbone conformation of the outer vestibule does not change significantly between different functional states but that local dynamics change significantly, underlining the importance of the hydrogen bond network behind the selectivity filter and the microscopic observables (e.g., dynamics of hydration) in K+ channel gating and C-type inactivation. 相似文献
12.
Membrane binding of the pH sensitive antimicrobial peptide LAH4 studied by EPR spectroscopy
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Peptide-membrane binding is vital for many biological events, including the bacteria combating by antimicrobial peptides. Using the pH sensitive LAH4 peptide as model, we employed a convenient electron paramagnetic resonance (EPR) method to study the peptide-membrane binding process in artificial phospholipid membranes. Based on spectral changes of the nitroxide radicallabeled to the peptides, we characterized binding kinetics and affinity of peptides to different phospholipid membranes. The binding affinity of LAH4 towards POPG was more than an order of magnitude higher than those towards DMPC and POPC. The binding kinetics showed that LAH4 initially bound to POPG much more quickly than to DMPC and POPC. Additionally, pH also affected the binding kinetics in LAH4-membrane interactions, which helped explain the pH dependent antimicrobial activity of LAH4. The method might be further used to monitor the membrane binding/cell penetration of antimicrobial peptide in living cells. 相似文献
13.
Lukoyanov D Yang ZY Barney BM Dean DR Seefeldt LC Hoffman BM 《Proceedings of the National Academy of Sciences of the United States of America》2012,109(15):5583-5587
Nitrogenase catalyzes the reduction of N2 and protons to yield two NH3 and one H2. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH3), or hydrazine as substrate recently was shown to trap a common intermediate, designated I. It was further concluded that the two N-atoms of N2 are hydrogenated alternately (“Alternating” (A) pathway). In the present work, Q-band CW EPR and 95Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state “non-Kramers” doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of 14,15N isotopologs plus 1,2H isotopologs of methyldiazene. It is concluded that: H has NH2 bound to FeMo-co and corresponds to the penultimate intermediate of N2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH3; I corresponds to the final intermediate in N2 reduction, the state formed after accumulation of eight electrons/protons, with NH3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the “prompt” alternating reaction pathway represents a draft mechanism for N2 reduction by nitrogenase. 相似文献
14.
15.
Bowen Qi Ayrianne J. Crawford Nicholas E. Wojtynek Megan B. Holmes Joshua J. Souchek Graca Almeida-Porada Quan P. Ly Samuel M. Cohen Michael A. Hollingsworth Aaron M. Mohs 《Nanomedicine : nanotechnology, biology, and medicine》2018,14(3):769-780
Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. In this study, hyaluronic acid derived nanoparticles with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG was not cytotoxic to healthy pancreatic epithelial cells and did not induce chemotaxis or phagocytosis, it accumulated significantly within the pancreas in an orthotopic pancreatic ductal adenocarcinoma model, and demonstrated contrast-enhancement for pancreatic lesions relative to non-diseased portions of the pancreas. Fluorescence microscopy showed higher fluorescence intensity in pancreatic lesions and splenic metastases due to NanoICG compared to ICG alone. The in vivo safety profile of NanoICG, including, biochemical, hematological, and pathological analysis of NanoICG-treated healthy mice, indicates negligible toxicity. These results suggest that NanoICG is a promising contrast agent for intraoperative detection of pancreatic tumors. 相似文献
16.
Sebastian Kallus Bernhard Englinger Julia Senkiv Anna Laemmerer Petra Heffeter Walter Berger Christian R. Kowol Bernhard K. Keppler 《Nanomedicine : nanotechnology, biology, and medicine》2018,14(8):2632-2643
Fibroblast growth factor receptor (FGFR) inhibitors like ponatinib and nintedanib are clinically approved for defined cancer patient cohorts but often exert dose-limiting adverse effects. Hence, we encapsulated the FGFR inhibitors ponatinib, PD173074, and nintedanib into polylactic acid nanoparticles and liposomes to enable increased tumor accumulation/specificity and reduce side effects. Different methods of drug loading were tested and the resulting formulations compared regarding average size distribution as well as encapsulation efficiency. Appropriate encapsulation levels were achieved for liposomal preparations only. Nanoencapsulation resulted in significantly decelerated uptake kinetics in vitro with clearly decreased short-term (up to 72?h) cytotoxicity at higher concentrations. However, in long-term clonogenic assays liposomal formations were equally or even more active as compared to the free drugs. Accordingly, in an FGFR inhibitor-sensitive murine osteosarcoma transplantation model (K7M2), only liposomal but not free ponatinib resulted in significant tumor growth inhibition (by 60.4%) at markedly reduced side effects. 相似文献
17.
Dinesh K. Mishra Ruchita Shandilya Pradyumna K. Mishra 《Nanomedicine : nanotechnology, biology, and medicine》2018,14(7):2023-2050
Over the recent couple of decades, pharmaceutical field has embarked most phenomenal noteworthy achievements in the field of medications as well as drug delivery. The rise of Nanotechnology in this field has reformed the existing drug delivery for targeting, diagnostic, remedial applications and patient monitoring. The convincing usage of nanotechnology in the conveyance of medications that prompts an extension of novel lipid-based nanocarriers and non-liposomal systems has been discussed. Present review deals with the late advances and updates in lipidic nanocarriers, their formulation strategies, challenging aspects, stability profile, clinical applications alongside commercially available products and products under clinical trials. This exploration may give a complete idea viewing the lipid based nanocarriers as a promising choice for the formulation of pharmaceutical products, the challenges looked by the translational process of lipid-based nanocarriers and the combating methodologies to guarantee the headway of these nanocarriers from bench to bedside. 相似文献
18.
John Weaver Yirong Yang Rebecca Purvis Theodore Weatherwax Gerald M. Rosen Ke Jian Liu 《Toxicology and applied pharmacology》2014
Abuse of methamphetamine (METH) is a major and significant societal problem in the US, as a number of studies have suggested that METH is associated with increased cerebrovascular events, hemorrhage or vasospasm. Although cellular and molecular mechanisms involved in METH-induced toxicity are not completely understood, changes in brain O2 may play an important role and contribute to METH-induced neurotoxicity including dopaminergic receptor degradation. Given that O2 is the terminal electron acceptor for many enzymes that are important in brain function, the impact of METH on brain tissue pO2in vivo remains largely uncharacterized. This study investigated striatal tissue pO2 changes in male C57BL/6 mice (16–20 g) following METH administration using EPR oximetry, a highly sensitive modality to measure pO2in vivo, in situ and in real time. We demonstrate that 20 min after a single injection of METH (8 mg/kg i.v.), the striatal pO2 was reduced to 81% of the pretreatment level and exposure to METH for 3 consecutive days further attenuated striatal pO2 to 64%. More importantly, pO2 did not recover fully to control levels even 24 h after administration of a single dose of METH and continual exposure to METH exacerbates the condition. We also show a reduction in cerebral blood flow associated with a decreased brain pO2 indicating an ischemic condition. Our findings suggests that administration of METH can attenuate brain tissue pO2, which may lead to hypoxic insult, thus a risk factor for METH-induced brain injury and the development of stroke in young adults. 相似文献
19.
Bozena Debska Ewa Spychaj-Fabisiak Wiesaw Szulc Renata Gaj Magdalena Banach-Szott 《Materials》2021,14(12)
The major indicator of soil fertility and productivity are humic acids (HAs) arising from decomposition of organic matter. The structure and properties of HAs depend, among others climate factors, on soil and anthropogenic factors, i.e., methods of soil management. The purpose of the research undertaken in this paper is to study humic acids resulting from the decomposition of crop residues of wheat (Triticum aestivum L.) and plant material of thuja (Thuja plicata D.Don.ex. Lamb) using electron paramagnetic resonance (EPR) spectroscopy. In the present paper, we report EPR studies carried out on two types of HAs extracted from forest soil and incubated samples of plant material (mixture of wheat straw and roots), both without soil and mixed with soil. EPR signals obtained from these samples were subjected to numerical analysis, which showed that the EPR spectra of each sample could be deconvoluted into Lorentzian and Gaussian components. It can be shown that the origin of HAs has a significant impact on the parameters of their EPR spectra. The parameters of EPR spectra of humic acids depend strongly on their origin. The HA samples isolated from forest soils are characterized by higher spin concentration and lower peak-to-peak width of EPR spectra in comparison to those of HAs incubated from plant material. 相似文献
20.
Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge
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Kim JM Altenbach C Kono M Oprian DD Hubbell WL Khorana HG 《Proceedings of the National Academy of Sciences of the United States of America》2004,101(34):12508-12513
The intramolecular interactions that stabilize the inactive conformation of rhodopsin are of primary importance in elucidating the mechanism of activation of this and other G protein-coupled receptors. In the present study, site-directed spin labeling is used to explore the role of a buried salt bridge between the protonated Schiff base at K296 in TM7 and its counterion at E113 in TM3. Spin-label sensors are placed at positions in the cytoplasmic surface of rhodopsin to monitor changes in the structure of the helix bundle caused by point mutations that disrupt the salt bridge. The single point mutations E113Q, G90D, and A292E, which were previously reported to cause constitutive activation of the apoprotein opsin, are found to cause profound movements of both TM3 and TM6 in the dark state, the latter of which is similar to that caused by light activation. The mutant M257Y, which constitutively activates opsin but does not disrupt the salt bridge, is shown to cause related but distinguishable structural changes. The double mutants E113Q/M257Y and G90D/M257Y produce strong activation of the receptor in the dark state. In the E113Q/M257Y mutant investigated with spin labeling, the movement of TM6 and other changes are exaggerated relative to either E113Q or M257Y alone. Collectively, the results provide structural evidence that the salt bridge is a key constraint maintaining the resting state of the receptor, and that the disruption of the salt bridge is the cause, rather than a consequence, of the TM6 motion that occurs upon activation. 相似文献