共查询到20条相似文献,搜索用时 15 毫秒
1.
Müller DJ Dencher NA Meier T Dimroth P Suda K Stahlberg H Engel A Seelert H Matthey U 《FEBS letters》2001,504(3):219-222
Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions. 相似文献
2.
Müller V 《Journal of bioenergetics and biomembranes》2004,36(1):115-125
The motor domain of A1A0 ATPases is composed of only two subunits, the stator subunit I and the rotor subunit c. Recent studies on the molecular biology of the A0 domains revealed the surprising finding that the gene encoding subunit c underwent several multiplication events leading to rotor subunits comprising 2, 3, or even 13 hairpin domains suggesting multimeric in different stoichiometry as well as monomeric rotors. The number of ion translocating groups per rotor ranges from 13 to 6. Furthermore, as deduced from the gene sequences H(+)-as well as Na(+)-driven rotors are found in archaea. Features previously thought to be distinctive for A0, F0 or V0 are all found in A0 suggesting that the differences encountered in the three classes of ATPases today emerged already very early in evolution. The extraordinary features and exceptional structural and functional variability in the rotor of A1A0 ATPases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life. 相似文献
3.
Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography. 相似文献
4.
Heymann JB Müller DJ Landau EM Rosenbusch JP Pebay-Peyroula E Büldt G Engel A 《Journal of structural biology》1999,128(3):243-249
The preponderance of structural data of the purple membrane from X-ray diffraction (XRD), electron crystallography (EC), and atomic force microscopy (AFM) allows us to ask questions about the structure of bacteriorhodopsin itself, as well as about the information derived from the different techniques. The transmembrane helices of bacteriorhodopsin are quite similar in both EC and XRD models. In contrast, the loops at the surfaces of the purple membrane show the highest variability between the atomic models, comparable to the height variance measured by AFM. The excellent agreement of the AFM topographs with the atomic models from XRD builds confidence in the results. Small technical difficulties in EC lead to poorer resolution of the loop structures, although the combination of atomic models with AFM surfaces allows clear interpretation of the extent and flexibility of the loop structures. While XRD remains the premier technique to determine very-high-resolution structures, EC offers a method to determine loop structures unhindered by three-dimensional crystal contacts, and AFM provides information about surface structures and their flexibility under physiological conditions. 相似文献
5.
Stahlberg H Müller DJ Suda K Fotiadis D Engel A Meier T Matthey U Dimroth P 《EMBO reports》2001,2(3):229-233
Synthesis of adenosine triphosphate (ATP) by the F1F0 ATP synthase involves a membrane-embedded rotary engine, the F0 domain, which drives the extra-membranous catalytic F1 domain. The F0 domain consists of subunits a1b2 and a cylindrical rotor assembled from 9–14 α-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits. 相似文献
6.
Susan M. Howitt Andrew J. W. Rodgers Lyndall P. Hatch Frank Gibson Graeme B. Cox 《Journal of bioenergetics and biomembranes》1996,28(5):415-420
F0F1-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F0F1-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed. 相似文献
7.
Glycerol facilitator (GF) is a tetrameric membrane protein responsible for the selective permeation of glycerol and water. Each of the four GF subunits forms a transmembrane channel. Every subunit consists of six helices that completely span the lipid bilayer, as well as two half-helices (TM7 and TM3). X-ray crystallography has revealed that the selectivity of GF is due to its unique amphipathic channel interior. To explore the structural dynamics of GF, we employ hydrogen/deuterium exchange (HDX) and oxidative labeling with mass spectrometry (MS). HDX-MS reveals that transmembrane helices are generally more protected than extramembrane segments, consistent with data previously obtained for other membrane proteins. Interestingly, TM7 does not follow this trend. Instead, this half-helix undergoes rapid deuteration, indicative of a highly dynamic local structure. The oxidative labeling behavior of most GF residues is consistent with the static crystal structure. However, the side chains of C134 and M237 undergo labeling although they should be inaccessible according to the X-ray structure. In agreement with our HDX-MS data, this observation attests to the fact that TM7 is only marginally stable. We propose that the highly mobile nature of TM7 aids in the efficient diffusion of guest molecules through the channel ("molecular lubrication"). In the absence of such dynamics, host-guest molecular recognition would favor semipermanent binding of molecules inside the channel, thereby impeding transport. The current work highlights the complementary nature of HDX, covalent labeling, and X-ray crystallography for the characterization of membrane proteins. 相似文献
8.
The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies [1]. The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor [2]. Phosphorylated CheY binds to isolated FliM [3] and may also interact with FliG [4], but its binding to basal bodies has not been measured. Using the chemorepellent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein-CheY fusion (GFP-CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy [6]. At acetate concentrations that cause solely clockwise rotation, GFP-CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits. GFP-CheY did not bind to rotors lacking FIiM/FliN, showing that these subunits are essential for the association. This assay provides a new means of monitoring protein-protein interactions in signal transduction pathways in living cells. 相似文献
9.
The heterotetrameric architecture of the epithelial sodium channel (ENaC). 总被引:19,自引:1,他引:18
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The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits. 相似文献
10.
11.
The structure, function and dynamics of photosystem two 总被引:6,自引:0,他引:6
One of the greatest challenges in modern photosynthesis research is to elucidate fully the structural and functional properties of photosystem two (PSII). This water-plasto-quinone oxidoreductase is located in a membrane complex composed of more than 25 subunits. The primary and secondary structures of all known subunits which constitute the central core of PSII are reviewed. How these subunits interact with each other to produce the tertiary and quaternary structure of PSII in vivo is not fully understood. However, electron microscopy is helping to fill this gap in our knowledge both by single particle analysis and electron crystallography. These studies suggest that active PSII is dimeric, although the functional significance of this oligomeric state is not yet understood. Moreover, the elucidation of the structure of photosystem one (PSI) by X-ray crystallography has revealed features which are likely to be relevant to PSII structure. It seems highly likely that the D1 protein with CP43 and D2 protein with CP47 (summing 11 transmembrane helices in each case) will have structural similarities to the organisation of PsaA and PsaB. It is likely that the turnover of the D1 protein is aided by the relatively easy removal of CP43 from this arrangement of the PSII core. 相似文献
12.
Structure of a voltage-dependent K+ channel beta subunit. 总被引:3,自引:0,他引:3
The integral membrane subunits of many voltage-dependent potassium channels are associated with an additional protein known as the beta subunit. One function of beta subunits is to modify K+ channel gating. We have determined the structure of the conserved core of mammalian beta subunits by X-ray crystallography at 2.8 A resolution. Like the integral membrane component of K+ channels, beta subunits form a four-fold symmetric structure. Each subunit is an oxidoreductase enzyme complete with a nicotinamide co-factor in its active site. Several structural features of the enzyme active site, including its location with respect to the four-fold axis, imply that it may interact directly or indirectly with the K+ channel's voltage sensor. This structure suggests a mechanism for coupling membrane electrical excitability directly to chemistry of the cell. 相似文献
13.
Masahiro Kitazawa Yoshihiro Kubo Koichi Nakajo 《The Journal of biological chemistry》2014,289(25):17597-17609
Kv4 is a voltage-gated K+ channel, which underlies somatodendritic subthreshold A-type current (ISA) and cardiac transient outward K+ (Ito) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K+ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits. 相似文献
14.
Fritz M Klyszejko AL Morgner N Vonck J Brutschy B Muller DJ Meier T Müller V 《The FEBS journal》2008,275(9):1999-2007
The Na(+) F(1)F(0) ATP synthase operon of the anaerobic, acetogenic bacterium Acetobacterium woodii is unique because it encodes two types of c subunits, two identical 8 kDa bacterial F(0)-like c subunits (c(2) and c(3)), with two transmembrane helices, and a 18 kDa eukaryal V(0)-like (c(1)) c subunit, with four transmembrane helices but only one binding site. To determine whether both types of rotor subunits are present in the same c ring, we have isolated and studied the composition of the c ring. High-resolution atomic force microscopy of 2D crystals revealed 11 domains, each corresponding to two transmembrane helices. A projection map derived from electron micrographs, calculated to 5 A resolution, revealed that each c ring contains two concentric, slightly staggered, packed rings, each composed of 11 densities, representing 22 transmembrane helices. The inner and outer diameters of the rings, measured at the density borders, are approximately 17 and 50 A. Mass determination by laser-induced liquid beam ion desorption provided evidence that the c rings contain both types of c subunits. The stoichiometry for c(2)/c(3) : c(1) was 9 : 1. Furthermore, this stoichiometry was independent of the carbon source of the growth medium. These analyses clearly demonstrate, for the first time, an F(0)-V(0) hybrid motor in an ATP synthase. 相似文献
15.
Clarke D Griffin S Beales L Gelais CS Burgess S Harris M Rowlands D 《The Journal of biological chemistry》2006,281(48):37057-37068
The p7 protein of hepatitis C virus functions as an ion channel both in vitro and in cell-based assays and is inhibited by amantadine, long alkyl chain imino-sugar derivatives, and amiloride compounds. Future drug design will be greatly aided by information on the stoichiometry and high resolution structure of p7 ion channel complexes. Here, we have refined a bacterial expression system for p7 based on a glutathione S-transferase fusion methodology that circumvents the inherent problems of hydrophobic protein purification and the limitations of chemical synthesis. Rotational averaging and harmonic analysis of transmission electron micrographs of glutathione S-transferase-FLAG-p7 fusion proteins in liposomes revealed a heptameric stoichiometry. The oligomerization of p7 protein was then confirmed by SDS-PAGE and mass spectrometry analysis of pure, concentrated FLAG-p7. The same protein was also confirmed to function as an ion channel in suspended lipid bilayers and was inhibited by amantadine. These data validate this system as a means of generating high resolution structural information on the p7 ion channel complex. 相似文献
16.
ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits—a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure. 相似文献
17.
Barber J 《Current opinion in structural biology》2002,12(4):523-530
A structure of photosystem II recently determined by X-ray crystallography at 3.8 A resolution complements structural studies using high-resolution electron microscopy and represents a major step towards understanding how photosynthetic organisms use light energy to oxidise water. 相似文献
18.
Cyclic nucleotide-gated (CNG) ion channels mediate the response to light in retinal rods. They are tetramers of two homologous subunits (alpha and beta), each of which is essential for the function of the channels in vivo. We have investigated the stoichiometry and arrangement of these two subunits to determine how they come together within an individual channel complex. We exploited the very specific geometric and spatial requirements for forming a high-affinity Ni2+-binding site to examine the number and relative positions of the subunits. We found that only an order of alpha/alpha/beta/beta could account qualitatively and quantitatively for the observed intersubunit coordination of Ni2+ in wild-type and mutant alpha/beta channels. Furthermore, our results suggest a structural dimerization among like subunits, at least at the level of the Ni2+-binding site. 相似文献
19.
Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains (VSDs) and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the VSDs control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the VSDs. 相似文献
20.
《生物化学与生物物理学报:生物膜》2022,1864(1):183809
Human aquaporin 10 (hAQP10) is an aquaglyceroporin that assists in maintaining glycerol flux in adipocytes during lipolysis at low pH. Hence, a molecular understanding of the pH-sensitive glycerol conductance may open up for drug development in obesity and metabolically related disorders. Control of hAQP10-mediated glycerol flux has been linked to the cytoplasmic end of the channel, where a unique loop is regulated by the protonation status of histidine 80 (H80). Here, we performed unbiased molecular dynamics simulations of three protonation states of H80 to unravel channel gating. Strikingly, at neutral pH, we identified a water coordination pattern with an inverted orientation of the water molecules in vicinity of the loop. Protonation of H80 results in a more hydrophobic loop conformation, causing loss of water coordination and leaving the pore often dehydrated. Our results indicate that the loss of such water interaction network may be integral for the destabilization of the loop in the closed configuration at low pH. Additionally, a residue unique to hAQP10 (F85) reveals structural importance by flipping into the channel in correlation with loop movements, indicating a loop-stabilizing role in the closed configuration. Taken together, our simulations suggest a unique gating mechanism combining complex interaction networks between water molecules and protein residues at the loop interface. Considering the role of hAQP10 in adipocytes, the detailed molecular insights of pH-regulation presented here will help to understand glycerol pathways in these cells and may assist in drug discovery for better management of human adiposity and obesity. 相似文献