Crystal Structures of a Group II Chaperonin Reveal the Open and Closed States Associated with the Protein Folding Cycle

Chaperonins are large protein complexes consisting of two stacked multisubunit rings, which open and close in an ATP-dependent manner to create a protected environment for protein folding. Here, we describe the first crystal structure of a group II chaperonin in an open conformation. We have obtained structures of the archaeal chaperonin from Methanococcus maripaludis in both a peptide acceptor (open) state and a protein folding (closed) state. In contrast with group I chaperonins, in which the equatorial domains share a similar conformation between the open and closed states and the largest motions occurs at the intermediate and apical domains, the three domains of the archaeal chaperonin subunit reorient as a single rigid body. The large rotation observed from the open state to the closed state results in a 65% decrease of the folding chamber volume and creates a highly hydrophilic surface inside the cage. These results suggest a completely distinct closing mechanism in the group II chaperonins as compared with the group I chaperonins.     

Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.     

Disposable flow through slide chamber

An inexpensive disposable flow through slide chamber using double face tape is described. The chamber allows observation of the free movement and interaction of microscopic invertebrates and has sufficient chamber volume to sustain life for adequate study. It further has a flow through capacity allowing observation of microscopic invertebrates and cells in continuously changing environments.     

新生大鼠大脑皮层神经元胞体ATP激活的离子通道及其特性

用膜片箝技术的细胞贴附式和内面向外式,在机械分离的新生ＳＤ大鼠的大脑皮层神经元上,记录到ＡＴＰ激活的离子通道。此通道的电导为３２ｐＳ,对Ｎａ~＋,Ｋ~＋和Ｃｓ~＋无选择性通透,而对Ｃｌ~－不通透。通道开放时间分布直方图多数需用双指数拟合,少数可用单指数拟合;通道关闭时间分布直方图均需用双指数拟合。通道的平均开放时间和开放概率均不依赖于膜电位;但通道的开放概率随着激动剂ＡＴＰ浓度的增加而增大。当电极内液无ＡＴＰ时,无通道电流。六烃季胶和美加明不能阻断此通道。上述结果表明,新生大鼠的大脑皮层神经元胞体可能存在ＡＴＰ激活的离子通道。     

Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.     

Interaction of human DNA topoisomerase II alpha with DNA: quantification by surface plasmon resonance

DNA topoisomerase II is an ATP-operated clamp that effects topological changes by capturing a double-stranded DNA segment and transporting it through another duplex. Surface plasmon resonance (SPR) was used to characterize interactions of human topoisomerase II alpha with different topological forms of DNA. Using a linear fragment of pUC18 DNA, the equilibrium binding constant of topoisomerase II alpha was determined to be 0.16 nM. The affinity was not affected by the absence of ATP or the presence of the bisdioxopiperazine catalytic inhibitor ICRF-187. Besides, similar affinities were found for several bisdioxopiperazine-resistant mutant enzymes. These results suggest that the mechanism of topoisomerase II alpha inhibition by ICRF-187 and its resistance does not directly involve the interaction of DNA with the enzyme. SPR was also adapted to measure levels of the closed clamp form of topoisomerase II present on DNA. As expected, a stable closed clamp form of the enzyme was detectable on circular DNA but not on linear DNA. Detection of the closed clamp required the presence of ATP and a bisdioxopiperazine, or a non-hydrolyzable analogue of ATP. In the presence of ATP and ICRF-187, several bisdioxopiperazine-resistant mutant enzymes failed to form detectable levels of stable closed clamp. Interestingly, a mutant of human topoisomerase II alpha with an altered active site tyrosine showed lower levels of closed clamp formation. In conclusion, SPR is able to (1) determine the kinetics of topoisomerase II with its DNA substrate and (2) quantify the enzyme's closed clamp formation under varying circumstances.     

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series : a depth prefilter over a 1·2 μm filter ; a 0·8 μm prefilter over a 0·45 μm filter ; and a 0·2 μm sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.     

Crystal structure of group II chaperonin in the open state

Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to a lack of high-resolution structure in the open state. Here, we report the first complete crystal structure of thermosome (rATcpnβ) in the open state from Acidianus tengchongensis. There is a ～30° rotation of the apical and lid domains compared with the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain, and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpnβ were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from the open to the closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.     

On the solution structure of the T4 sliding clamp (gp45)

Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces. This is in contrast to what is observed in the X-ray crystal structure. The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A. This leads to the conclusion that gp45 exists in an asymmetric open state in solution. The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface. The ramifications for replisome remodeling by this pathway are discussed.     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

Quantification of circular DNA breakage by fluorescence scanning of ethidium bromide-stained horizontal gels: application to the effect of intercalating agents

Direct scanning of the fluorescence of DNA in ethidium bromide-stained agarose gels allowed the quantification of closed and open circular DNA forms. Fluorescence of form I was higher than expected compared to form II. Application of this technique is shown for an intercalating drug treatment of DNA.     

A long-term stable and adjustable osmotic pump for small volume flow based on principles of phloem loading

An adjustable pump for microfluidics employing principles of osmoregulation analogous to those of phloem loading in plant leaves has been constructed and tested. Volume flow arises in a hollow fibre with vapour-permeable hydrophobic membrane. The fibre is connected to a source chamber filled with salt crystals and saturated salt solution. The source chamber takes up water through a relatively small membrane area and delivers saturated salt solution to one end of the capillary flow path within the hollow fibre. A stationary osmotic gradient is sustained in the hollow fibre lumen by constant input of saturated salt solution and radial osmotic water absorption. The strong temperature dependence of isothermal membrane distillation enables adjustment of the flow rate up to 20 nL/s. The pump provides pulse-free flow of any liquid with constant rate for at least 26 days without recharging the source chamber. Backpressures up to 1 bar decrease the flow rate by less than 4%. The volume delivered at a constant rate is more than 40 times larger than the volume of the source chamber. Osmoregulatory pumps of the described type may be useful for microinfusion, microdialysis and analytical microsystems.     

A non-viral gene delivery system designed for clinical use

Gene delivery can be accomplished using non-viral systems, and these have received increased attention These include ex vivo transfection of cells using an electric field to induce transient cell-membrane permeability (electroporation). This approach has the distinct advantage of not requiring the inclusion of a secondary agent (e.g. a lipid, viral package or carrier protein) any of which can be immunogenic or toxic. Available electroporation systems utilize a low volume (<1 mL) processing chamber and are open systems. The MaxCyte system employs a continuous flow design and can very rapidly process volumes ranging from 0.02 mL to >1 L. Transgenes for markers (eGFP) and functional proteins (e.g., cytokines, angiogenic factors) have been loaded in plasmids up to 14 kB in size. With appropriate application of pre- and post-processing cell manipulations, very satisfactory loading efficiencies and cell viability have been obtained. Cells can be processed with multiple plasmids, resulting in expression of the corresponding number of gene products. This capability has been considered for therapeutic and bioprocessing applications. The MaxCyte system was designed specifically for ex vivo clinical applications. The electrodes are manufactured of special materials and under precise conditions, in order to eliminate potential risks from electrolytic effects. The processing chamber and associated containers can be provided as disposable, sterile, closed (or functionally closed) systems-quite similar to the disposable harnesses used with cell separators. This system is thus suitable for integration into a current good manufacturing practice environment.     

Three conformations of an archaeal chaperonin, TF55 from Sulfolobus shibatae

Chaperonins are cylindrical, oligomeric complexes, essential for viability and required for the folding of other proteins. The GroE (group I) subfamily, found in eubacteria, mitochondria and chloroplasts, have 7-fold symmetry and provide an enclosed chamber for protein subunit folding. The central cavity is transiently closed by interaction with the co-protein, GroES. The most prominent feature specific to the group II subfamily, found in archaea and in the eukaryotic cytosol, is a long insertion in the substrate-binding region. In the archaeal complex, this forms an extended structure acting as a built-in lid, obviating the need for a GroES-like co-factor. This extension occludes a site known to bind non-native polypeptides in GroEL. The site and nature of substrate interaction are not known for the group II subfamily. The atomic structure of the thermosome, an archaeal group II chaperonin, has been determined in a fully closed form, but the entry and exit of protein substrates requires transient opening. Although an open form has been investigated by electron microscopy, conformational changes in group II chaperonins are not well characterized. Using electron cryo-microscopy and three-dimensional reconstruction, we describe three conformations of a group II chaperonin, including an asymmetric, bullet-shaped form, revealing the range of domain movements in this subfamily.     

An Alternative Homodimerization Interface of MnmG Reveals a Conformational Dynamics that Is Essential for Its tRNA Modification Function

The Escherichia coli homodimeric proteins MnmE and MnmG form a functional complex, MnmEG, that modifies tRNAs using GTP, methylene-tetrahydrofolate, FAD, and glycine or ammonium. MnmE is a tetrahydrofolate- and GTP-binding protein, whereas MnmG is a FAD-binding protein with each protomer composed of the FAD-binding domain, two insertion domains, and the helical C-terminal domain. The detailed mechanism of the MnmEG-mediated reaction remains unclear partially due to incomplete structural information on the free- and substrate-bound forms of the complex. In this study, we show that MnmG can adopt in solution a dimer arrangement (form I) different from that currently considered as the only biologically active (form II). Normal mode analysis indicates that form I can oscillate in a range of open and closed conformations. Using isothermal titration calorimetry and native red electrophoresis, we show that a form-I open conformation, which can be stabilized in vitro by the formation of an interprotomer disulfide bond between the catalytic C277 residues, appears to be involved in the assembly of the MnmEG catalytic center. We also show that residues R196, D253, R436, R554 and E585 are important for the stabilization of form I and the tRNA modification function. We propose that the form I dynamics regulates the alternative access of MnmE and tRNA to the MnmG FAD active site. Finally, we show that the C-terminal region of MnmG contains a sterile alpha motif domain responsible for tRNA–protein and protein–protein interactions.     

Molecular dynamics simulations reveal that apo‐HisJ can sample a closed conformation

The Escherichia coli histidine binding protein HisJ is a type II periplasmic binding protein (PBP) that preferentially binds histidine and interacts with its cytoplasmic membrane ABC transporter, HisQMP₂, to initiate histidine transport. HisJ is a bilobal protein where the larger Domain 1 is connected to the smaller Domain 2 via two linking strands. Type II PBPs are thought to undergo “Venus flytrap” movements where the protein is able to reversibly transition from an open to a closed conformation. To explore the accessibility of the closed conformation to the apo state of the protein, we performed a set of all‐atom molecular dynamics simulations of HisJ starting from four different conformations: apo‐open, apo‐closed, apo‐semiopen, and holo‐closed. The simulations reveal that the closed conformation is less dynamic than the open one. HisJ experienced closing motions and explored semiopen conformations that reverted to closed forms resembling those found in the holo‐closed state. Essential dynamics analysis of the simulations identified domain closing/opening and twisting as main motions. The formation of specific inter‐hinge strand and interdomain polar interactions contributed to the adoption of the closed apo‐conformations although they are up to 2.5‐fold less prevalent compared with the holo‐closed simulations. The overall sampling of the closed form by apo‐HisJ provides a rationale for the binding of unliganded PBPs with their cytoplasmic membrane ABC transporters. Proteins 2014; 82:386–398. © 2013 Wiley Periodicals, Inc.     

Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium.

The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and II) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and II resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosome-like structures. Peaks I and II were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosome structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.     

Mechanical simulator of the cardiovascular system

To devise and to build a mechanical simulator of the cardiovascular system of increasing complexity is a fascinating experience for a medical Physicist. We did it, and the effort to match the solutions with the objectives forced us to deepen the knowledge of the physiological aspects, to devise different solutions and to compare their results. This paper describes the final solution and shows the results, discussing the theoretical and practical aspects of the different choices.The ventricle is simulated by a pumping syringe with an external pulsing chamber to accomplish the Frank–Starling mechanism; the coronary circulation by a nonlinear hydraulic resistance device; the aorta by different wall thickness rubber tubes; the arterial vascular resistance by a thin, variable length tube; the venous reservoir by a variable volume chamber connected to a reservoir simulating the atrium.The simulator was mainly devoted to teaching purposes, but the possibility to modify the mechanical characteristics of the single components moved it to be used also for research, with an unexpected satisfaction.     

Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.     

Synthesis of two different molecular forms of ribulose-bisphosphate carboxylase/oxygenase in Rhodopseudomonas blastica grown in batch culture

The expression of the two different molecular forms (form I and form II) of ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) in Rhodopseudomonas blastica during growth in batch on pyruvate–malate medium was investigated. During the early exponential phase of growth, only form I enzyme was synthesized. At the mid-exponential phase of growth, both forms were expressed, although form I enzyme was predominant. At the late exponential phase, form I and form II enzymes were synthesized but form II enzyme predominated. It is concluded that form I and form II RuBisCO enzymes in R. blastica are differentially expressed and this may be mediated by the level of CO2 in the growth medium.