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1.
    
The study of ion channels dates back to the 1950s and the groundbreaking electrophysiology work of Hodgin and Huxley, who used giant squid axons to probe how action potentials in neurons were initiated and propagated. More recently, several experiments using different structural biology techniques and approaches have been conducted to try to understand how potassium ions permeate through the selectivity filter of potassium ion channels. Two mechanisms of permeation have been proposed, and each of the two mechanisms is supported by different experiments. The key structural biology experiments conducted so far to try to understand how ion permeation takes place in potassium ion channels are reviewed and discussed. Protein crystallography has made, and continues to make, key contributions in this field, often through the use of anomalous scattering. Other structural biology techniques used to study the contents of the selectivity filter include solid‐state nuclear magnetic resonance and two‐dimensional infrared spectroscopy, both of which make clever use of isotopic labeling techniques, while molecular‐dynamics simulations of ion flow through the selectivity filter have been enabled by the growing number of potassium ion channel structures deposited in the Protein Data Bank.  相似文献   

2.
The polymerization of laminin into a cell-associated network--a key step in basement membrane assembly--is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN-LE1-2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network.  相似文献   

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Trehalose‐6‐phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6‐phosphate (T6P) to yield glucose and glucose 6‐phosphate. The products of this reaction can be further metabolized by the energy‐generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p‐nitrophenyl‐α‐d ‐glucopyranoside (R201Q–pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo‐1,6‐glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX‐5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the 281HHLK284 motif and Lys292 play critical roles in substrate discrimination by BlTreA.  相似文献   

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We determined the three-dimensional (3D) crystal structure of protein TM841, a protein product from a hypothetical open-reading frame in the genome of the hyperthermophile bacterium Thermotoga maritima, to 2.0 A resolution. The protein belongs to a large protein family, DegV or COG1307 of unknown function. The 35 kDa protein consists of two separate domains, with low-level structural resemblance to domains from other proteins with known 3D structures. These structural homologies, however, provided no clues for the function of TM841. But the electron density maps revealed clear density for a bound fatty-acid molecule in a pocket between the two protein domains. The structure indicates that TM841 has the molecular function of fatty-acid binding and may play a role in the cellular functions of fatty acid transport or metabolism.  相似文献   

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The locations of H atoms in biological structures can be difficult to determine using X‐ray diffraction methods. Neutron diffraction offers a relatively greater scattering magnitude from H and D atoms. Here, 1.65 Å resolution neutron diffraction studies of fully perdeuterated and selectively CH3‐protonated perdeuterated crystals of Pyrococcus furiosus rubredoxin (D‐rubredoxin and HD‐rubredoxin, respectively) at room temperature (RT) are described, as well as 1.1 Å resolution X‐ray diffraction studies of the same protein at both RT and 100 K. The two techniques are quantitatively compared in terms of their power to directly provide atomic positions for D atoms and analyze the role played by atomic thermal motion by computing the σ level at the D‐atom coordinate in simulated‐annealing composite D‐OMIT maps. It is shown that 1.65 Å resolution RT neutron data for perdeuterated rubredoxin are ∼8 times more likely overall to provide high‐confidence positions for D atoms than 1.1 Å resolution X‐ray data at 100 K or RT. At or above the 1.0σ level, the joint X‐ray/neutron (XN) structures define 342/378 (90%) and 291/365 (80%) of the D‐atom positions for D‐rubredoxin and HD‐rubredoxin, respectively. The X‐ray‐only 1.1 Å resolution 100 K structures determine only 19/388 (5%) and 8/388 (2%) of the D‐atom positions above the 1.0σ level for D‐rubredoxin and HD‐rubredoxin, respectively. Furthermore, the improved model obtained from joint XN refinement yielded improved electron‐density maps, permitting the location of more D atoms than electron‐density maps from models refined against X‐ray data only.  相似文献   

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Deltarhodopsin, a new member of the microbial rhodopsin family, functions as a light‐driven proton pump. Here, we report the three‐dimensional structure of deltarhodopsin (dR3) from Haloterrigena thermotolerans at 2.7 Å resolution. A crystal belonging to space group R32 (a, b = 111.71 Å, c = 198.25 Å) was obtained by the membrane fusion method. In this crystal, dR3 forms a trimeric structure as observed for bacteriorhodopsin (bR). Structural comparison of dR with bR showed that the inner part (the proton release and uptake pathways) is highly conserved. Meanwhile, residues in the protein–protein contact region are largely altered so that the diameter of the trimeric structure at the cytoplasmic side is noticeably larger in dR3. Unlike bR, dR3 possesses a helical segment at the C‐terminal region that fills the space between the AB and EF loops. A significant difference is also seen in the FG loop, which is one residue longer in dR3. Another peculiar property of dR3 is a highly crowded distribution of positively charged residues on the cytoplasmic surface, which may be relevant to a specific interaction with some cytoplasmic component.Proteins 2013; © 2013 Wiley Periodicals, Inc.  相似文献   

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The mechanism by which potassium ions are transported through ion channels is currently being investigated by several groups using many different techniques. Clarification of the location of water molecules during transport is central to understanding how these integral membrane proteins function. Neutrons have a unique sensitivity to both hydrogen and potassium, rendering neutron crystallography capable of distinguishing waters from K+ ions. Here, the collection of a complete neutron data set from a potassium ion channel to a resolution of 3.55 Å using the Macromolecular Neutron Diffractometer (MaNDi) is reported. A room‐temperature X‐ray data set was also collected from the same crystal to a resolution of 2.50 Å. Upon further refinement, these results will help to further clarify the ion/water population within the selectivity filter of potassium ion channels.  相似文献   

11.
    
In a crystallography experiment, a crystal is irradiated with X-rays whose diffracted waves are collected and measured. The reconstruction of the structure of the molecule in the crystal requires knowledge of the phase of the diffracted waves, information that is lost in the passage from the three-dimensional structure of the molecule to its diffraction pattern. It can be recovered using experimental methods such as heavy-atom isomorphous replacement and anomalous scattering or by molecular replacement, which relies on the availability of an atomic model of the target structure. This can be the structure of the target protein itself, if a previous structure determination is available, or a computational model or, in some cases, the structure of a homologous protein. It is not straightforward to predict beforehand whether or not a computational model will work in a molecular replacement experiment, although some rules of thumb exist. The consensus is that even minor differences in the quality of the model, which are rather difficult to estimate a priori, can have a significant effect on the outcome of the procedure. We describe here a method for quickly assessing whether a protein structure can be solved by molecular replacement. The procedure consists in submitting the sequence of the target protein to a selected list of freely available structure prediction servers, cluster the resulting models, select the representative structures of each cluster and use them as search models in an automatic phasing procedure. We tested the procedure using the structure factors of newly released proteins of known structure downloaded from the Protein Data Bank as soon as they were made available. Using our automatic procedure we were able to obtain an interpretable electron density map in more than half the cases.  相似文献   

12.
    
Chang Q  Yan XX  Gu SY  Liu JF  Liang DC 《Proteins》2008,73(1):254-258
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A novel vitreous carbon mount for macromolecular crystallography, suitable for neutron and X‐ray crystallographic studies, has been developed. The technology described here is compatible both with X‐ray and neutron cryo‐crystallography. The mounts have low density and low background scattering for both neutrons and X‐rays. They are prepared by laser cutting, allowing high standards of production quality, the ability to custom‐design the mount to specific crystal sizes and large‐scale production.  相似文献   

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Shigella dysentriae and other Gram‐negative human pathogens are able to use iron from heme bound to hemoglobin for growing. We solved at 2.6 Å resolution the 3D structure of the TonB‐dependent heme/hemoglobin outer membrane receptor ShuA from S. dysenteriae. ShuA binds to hemoglobin and transports heme across the outer membrane. The structure consists of a C‐terminal domain that folds into a 22‐stranded transmembrane β‐barrel, which is filled by the N‐terminal plug domain. One distal histidine ligand of heme is located at the apex of the plug, exposed to the solvent. His86 is situated 9.86 Å apart from His420, the second histidine involved in the heme binding. His420 is in the extracellular loop L7. The heme coordination by His86 and His420 involves conformational changes. The comparisons with the hemophore receptor HasR of Serratia marcescens bound to HasA‐Heme suggest an extracellular induced fit mechanism for the heme binding. The loop L7 contains hydrophobic residues which could interact with the hydrophobic porphyring ring of heme. The energy required for the transport by ShuA is derived from the proton motive force after interactions between the periplasmic N‐terminal TonB‐box of ShuA and the inner membrane protein, TonB. In ShuA, the TonB‐box is buried and cannot interact with TonB. The structural comparisons with HasR suggest its conformational change upon the heme binding for interacting with TonB. The signaling of the heme binding could involve a hydrogen bond network going from His86 to the TonB‐box. Proteins 2010. © 2009 Wiley‐Liss, Inc.  相似文献   

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Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co‐infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of NgCt co‐infections. Development of a safe, effective, and inexpensive dual therapy for NgCt co‐infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X‐ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high‐throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.  相似文献   

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The proteins Ctf19, Okp1, Mcm21 and Ame1 are the components of COMA, a subassembly of budding-yeast kinetochores. We have determined the crystal structure of a conserved COMA subcomplex--the Ctf19-Mcm21 heterodimer--from Kluyveromyces lactis. Both proteins contain 'double-RWD' domains, which together form a Y-shaped framework with flexible N-terminal extensions. The kinetochore proteins Csm1, Spc24 and Spc25 have related single RWD domains, and Ctf19 and Mcm21 associate with pseudo-twofold symmetry analogous to that in the Csm1 homodimer and the Spc24-Spc25 heterodimer. The double-RWD domain core of the Ctf19-Mcm21 heterodimer is sufficient for association with Okp1-Ame1; the less conserved N-terminal regions may interact with components of a more extensive 'CTF19 complex'. Our structure shows the RWD domain to be a recurring module of kinetochore architecture that may be present in other kinetochore substructures. Like many eukaryotic molecular machines, kinetochores may have evolved from simpler assemblies by multiplication of a few ancestral modules.  相似文献   

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Periplasmic heme‐binding proteins (PBPs) in Gram‐negative bacteria are components of the heme acquisition system. These proteins shuttle heme across the periplasmic space from outer membrane receptors to ATP‐binding cassette (ABC) heme importers located in the inner‐membrane. In the present study, we characterized the structures of PBPs found in the pathogen Burkholderia cenocepacia (BhuT) and in the thermophile Roseiflexus sp. RS‐1 (RhuT) in the heme‐free and heme‐bound forms. The conserved motif, in which a well‐conserved Tyr interacts with the nearby Arg coordinates on heme iron, was observed in both PBPs. The heme was recognized by its surroundings in a variety of manners including hydrophobic interactions and hydrogen bonds, which was confirmed by isothermal titration calorimetry. Furthermore, this study of 3 forms of BhuT allowed the first structural comparison and showed that the heme‐binding cleft of BhuT adopts an “open” state in the heme‐free and 2‐heme‐bound forms, and a “closed” state in the one‐heme‐bound form with unique conformational changes. Such a conformational change might adjust the interaction of the heme(s) with the residues in PBP and facilitate the transfer of the heme into the translocation channel of the importer.  相似文献   

20.
    
Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of ?483 ± 1 mV and ?187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.  相似文献   

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