Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Direct Observation of Structure and Dynamics of Photogenerated Charge Carriers in Poly(3-hexylthiophene) Films by Femtosecond Time-Resolved Near-IR Inverse Raman Spectroscopy.

The initial charge separation process of conjugated polymers is one of the key factors for understanding their conductivity. The structure of photogenerated transients in conjugated polymers can be observed by resonance Raman spectroscopy in the near-IR region because they exhibit characteristic low-energy transitions. Here, we investigate the structure and dynamics of photogenerated transients in a regioregular poly(3-hexylthiophene) (P3HT):[6,6]-phenyl-C61-butyric acid methyl ester (PCBM) blend film, as well as in a pristine P3HT film, using femtosecond time-resolved resonance inverse Raman spectroscopy in the near-IR region. The transient inverse Raman spectrum of the pristine P3HT film at 50 ps suggests coexistence of neutral and charged excitations, whereas that of the P3HT:PCBM blend film at 50 ps suggests formation of positive polarons with a different structure from those in an FeCl3-doped P3HT film. Time-resolved near-IR inverse Raman spectra of the blend film clearly show the absence of charge separation between P3HT and PCBM within the instrument response time of our spectrometer, while they indicate two independent pathways of the polaron formation with time constants of 0.3 and 10 ps.

Temperature-Dependent Evolution of Raman Spectra of Methylammonium Lead Halide Perovskites, CH3NH3PbX3 (X = I, Br).

We present a Raman study on the phase transitions of organic/inorganic hybrid perovskite materials, CH3NH3PbX3 (X = I, Br), which are used as solar cells with high power conversion efficiency. The temperature dependence of the Raman bands of CH3NH3PbX3 (X = I, Br) was measured in the temperature ranges of 290 to 100 K for CH3NH3PbBr3 and 340 to 110 K for CH3NH3PbI3. Broad ν1 bands at ~326 cm-1 for MAPbBr3 and at ~240 cm-1 for MAPbI3 were assigned to the MA⁻PbX3 cage vibrations. These bands exhibited anomalous temperature dependence, which was attributable to motional narrowing originating from fast changes between the orientational states of CH3NH3⁺ in the cage. Phase transitions were characterized by changes in the bandwidths and peak positions of the MA⁻cage vibration and some bands associated with the NH3⁺ group.

Insight into adaptive remodeling of the rotor ring complex of the bacterial flagellar motor.

The bacterial flagellar motor rotates in both counterclockwise (CCW) and clockwise (CW) directions. FliG, FliM and FliN form the C ring on the cytoplasmic face of the MS ring made of a transmembrane protein, FliF. The C ring acts not only as a rotor but also as a switch of the direction of motor rotation. FliG consists of three domains: FliGN, FliGM and FliGC. FliGN directly binds to FliF. Intermolecular interactions between FliGM and FliGC drive FliG ring formation. FliGM is responsible for the interaction with FliM. FliGC is involved in the interaction with the stator protein MotA. Adaptive remodeling of the C ring occurs when the motor switches between the CCW and CW states. However, it remained unknown how. Here, we report the effects of a CW-locked deletion mutation (ΔPEV) in FliG of Thermotaoga maritia (Tm-FliG) on FliG-FliG and FliG-FliM interactions. The PEV deletion stabilized the intramolecular interaction between FliGM and FliGC, thereby suppressing the oligomerization of Tm-FliGMC in solution. This deletion also induced a conformational change of HelixMC connecting FliGM and FliGC to reduce the binding affinity of Tm-FliGMC for FliM. We will discuss adaptive remodeling of the C ring responsible for flagellar motor switching.

Rearrangements of α-helical structures of FlgN chaperone control the binding affinity for its cognate substrates during flagellar type III export.

The bacterial flagellar type III export chaperones not only act as bodyguards to protect their cognate substrates from aggregation and proteolysis in the cytoplasm but also ensure the order of export through their interactions with an export gate protein FlhA. FlgN chaperone binds to FlgK and FlgL with nanomolar affinity and transfers them to FlhA for their efficient and rapid transport for the formation of the hook-filament junction zone. However, it remains unknown how FlgN releases FlgK and FlgL at the FlhA export gate platform in a timely manner. Here, we have solved the crystal structure of Salmonella FlgN at 2.3 Å resolution and carried out structure-based functional analyses. FlgN consists of three α helices, α1, α2 and α3. Helix α1 adopts two distinct, extended and bent conformations through the conformational change of N-loop between α1 and α2. The N-loop deletion not only increases the probability of FlgN dimer formation but also abolish the interaction between FlgN and FlgK. Highly conserved Asn-92, Asn-95 and Ile-103 residues in helix α3 are involved in the strong interaction with FlgK. We propose that the N-loop coordinates helical rearrangements of FlgN with the association and dissociation of its cognate substrates during their export.

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone.

FliS chaperone binds to flagellin FliC in the cytoplasm and transfers FliC to a sorting platform of the flagellar type III export apparatus through the interaction between FliS and FlhA for rapid and efficient protein export during flagellar filament assembly. FliS also suppresses the secretion of an anti-σ factor, FlgM. Loss of FliS results in a short filament phenotype although the expression levels of FliC are increased considerably due to an increase in the secretion level of FlgM. Here to clarify the rate limiting step of FliC export in the absence of FliS, we isolated bypass mutants from a Salmonella ΔfliS mutant. All the bypass mutations were identified in FliC. These bypass mutations increased the export rate of FliC by ca. twofold, allowing the bypass mutant cells to produce longer filaments than the parental ΔfliS cells. Both far-UV CD measurements and limited proteolysis revealed that the bypass mutations significantly destabilize the folded structure of FliC monomer. These results suggest that an unfolding step of FliC limits the export rate of FliC in the ΔfliS mutant, thereby producing short filaments. We propose that FliS promotes FliC docking at the FlhA platform to facilitate subsequent unfolding of FliC.

Structural Study of MPN387, an Essential Protein for Gliding Motility of a Human-Pathogenic Bacterium, Mycoplasma pneumoniae.

UNLABELLED: Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed. IMPORTANCE: Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae This tiny, 2-µm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma "gliding motility" is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism.

Importance of the monomer-dimer-tetramer interconversion of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock oscillator consists of three proteins, KaiA, KaiB and KaiC, which interact with each other to generate circadian oscillations in the presence of ATP (the in vitro KaiABC clock oscillator). KaiB comprises four subunits organized as a dimer of dimers. Our previous study suggested that, on interaction with KaiC, the tetrameric KaiB molecule dissociates into two molecules of dimeric KaiB. It is uncertain whether KaiB also exists as a monomer and whether the KaiB monomer can drive normal circadian oscillation. To address these questions, we constructed a new KaiB oligomer mutant with an N-terminal deletion, KaiB10-108 . KaiB10-108 was a monomer at 4 °C but a dimer at 35 °C. KaiB10-108 was able to drive normal clock oscillation in an in vitro reconstituted KaiABC clock oscillator at 25 °C, but it was not able to drive normal circadian gene expression rhythms in cyanobacterial cells at 41 °C. Wild-type KaiB existed in equilibrium between a dimer and tetramer at lower KaiB concentrations or in the presence of 1 m NaCl. Our findings suggest that KaiB is in equilibrium between a monomer, dimer and tetramer in cyanobacterial cells.

Purification of hepatitis C virus core protein in non-denaturing condition.

Hepatitis C virus (HCV) is the major cause of chronic hepatitis and hepatocellular carcinoma. Among its structural proteins, the HCV core protein has been implicated in liver disease. Understanding the role of HCV core proteins in viral diseases is crucial to elucidating disease mechanisms and identifying potential drug targets. However, purification challenges hinder the comprehensive elucidation of the structure and biochemical properties of HCV core proteins. In this study, we successfully solubilized bacterially expressed core protein using a high-salt and detergent-containing buffer and bypassed the denaturing-refolding process. Size-exclusion chromatography revealed three distinct peaks in the HCV-infected cell lysate, with the bacterially expressed soluble core protein corresponding to its second peak. Using a combination of affinity, size exclusion, and multi-modal chromatography purification techniques, we achieved a purity of > 95% for the core protein. Analytical ultracentrifugation revealed monomer formation in the solution. Far UV Circular dichroism spectroscopy identified 25.53% alpha helices and 20.26% beta sheets. These findings strongly suggest that the purified core proteins retained one of the native structures observed in HCV-infected cells.

The roles of the dimeric and tetrameric structures of the clock protein KaiB in the generation of circadian oscillations in cyanobacteria.

The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94).

Phase-dependent generation and transmission of time information by the KaiABC circadian clock oscillator through SasA-KaiC interaction in cyanobacteria.

Circadian clocks allow organisms to predict environmental changes of the day/night cycle. In the cyanobacterial circadian clock machinery, the phosphorylation level and ATPase activity of the clock protein KaiC oscillate with a period of approximately 24 h. The time information is transmitted from KaiC to the histidine kinase SasA through the SasA autophosphorylation-enhancing activity of KaiC, ultimately resulting in genome-wide transcription cycles. Here, we showed that SasA derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 has the domain structure of an orthodox histidine kinase and that its C-terminal domain, which contains a phosphorylation site at His160, is responsible for the autophosphorylation activity and the temperature- and phosphorylation state-dependent trimerization / hexamerization activity of SasA. SasA and KaiC associate through their N-terminal domains with an affinity that depends on their phosphorylation states. Furthermore, the SasA autophosphorylation-enhancing activity of KaiC requires the C-terminal ATPase catalytic site and depends on its phosphorylation state. We show that the phosphotransfer activity of SasA is essential for the generation of normal circadian gene expression in cyanobacterial cells. Numerical simulations suggest that circadian time information (free phosphorylated SasA) is released mainly by unphosphorylated KaiC during the late subjective night.

Structural insight into the regulatory mechanisms of interactions of the flagellar type III chaperone FliT with its binding partners.

For self-assembly of the bacterial flagellum, most of the flagellar component proteins synthesized in the cytoplasm are exported by the flagellar type III export apparatus to the growing, distal end. Flagellar protein export is highly organized and well controlled in every step of the flagellar assembly process. Flagellar-specific chaperones not only facilitate the export of their cognate proteins, as well as prevent their premature aggregation in the cytoplasm, but also play a role in fine-tuning flagellar gene expression to be coupled with the flagellar assembly process. FliT is a flagellar-specific chaperone responsible for the export of the filament-capping protein FliD and for negative control of flagellar gene expression by binding to the FlhDC complex. Here we report the crystal structure of Salmonella FliT at 3.2-A resolution. The structural and biochemical analyses clearly reveal that the C-terminal segment of FliT regulates its interactions with the FlhDC complex, FliI ATPase, and FliJ (subunits of the export apparatus), and that its conformational change is responsible for the switch in its binding partners during flagellar protein export.

Synthesis of kinetically stabilized 1,2-dihydrodisilenes.

Kinetically stabilized 1,2-dihydrodisilenes were successfully synthesized and isolated by the introduction of sterically protecting bulky aryl groups. These 1,2-dihydrodisilenes exhibit distinct Si═Si double-bond character in both solution and the solid state. The Si-H bonds in these 1,2-dihydrodisilenes exhibit higher s character than those of typical σ(4),λ(4)-hydrosilanes. Moderate heating of these 1,2-dihydrodisilenes in solution resulted in their isomerization to the corresponding trihydrodisilanes, with an intramolecular hydrogen migration as the rate-determining step.

Characterization of the periplasmic domain of MotB and implications for its role in the stator assembly of the bacterial flagellar motor.

MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotB(C)). MotB(C) significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotB(C)(Delta197-210), showed a significantly reduced inhibitory effect. Wild-type MotB(C) and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotB(C)-His(6) by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotB(C) heterodimer formation in the periplasm. However, the monomeric mutant variant MotB(C)(Delta197-210) did not bind to MotB, suggesting that MotB(C) is directly involved in stator assembly. We propose that the MotB(C) dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.

Synthesis and reactions of a stable 1,2-diaryl-1,2-dibromodisilene: a precursor for substituted disilenes and a 1,2-diaryldisilyne.

Synthesis and isolation of the stable diaryldibromodisilene, Bbt(Br)SiSi(Br)Bbt, has been accomplished for the first time. The dibromodisilene underwent substitution reactions with organometallic reagents on the low-coordinated silicon atom to afford the corresponding substituted disilenes. Furthermore, the reaction of 1 with t-BuLi afforded the corresponding 1,2-diaryldisilyne, BbtSi[triple bond]SiBbt, the characters of which were revealed by spectroscopic and crystallographic analyses.

Oligomerization of the bacterial flagellar ATPase FliI is controlled by its extreme N-terminal region.

Salmonella FliI is the flagellar ATPase which converts the energy of ATP hydrolysis into the export of flagellar proteins. It forms a ring-shaped oligomer in the presence of ATP, its analogs, or phospholipids. The extreme N-terminal region of FliI has an unstable conformation and is responsible for the interaction with other components of the export apparatus and for regulation of the catalytic mechanism. To understand the role of this N-terminal region in more detail, we used multi-angle light-scattering, analytical ultracentrifugation, far-UV CD and biochemical methods to characterize a partially functional variant of FliI, missing its first seven amino acid residues (His-FliI(Delta1-7)), whose ATPase activity is about ten times lower than that of wild-type FliI. His-FliI(Delta1-7) is monomeric in solution. The deletion increased the content of alpha-helix, suggesting that the deletion stabilizes the unstable N-terminal region into an alpha-helical conformation. The deletion did not influence the K(m) value for ATP. However, unlike the wild-type, ATP and acidic phospholipids did not induce oligomerization of His-FliI(Delta1-7) or increase its ATPase activity. These results suggest that the deletion suppresses the oligomerization of FliI, and that a conformational change in the unstable N-terminal region is required for FliI oligomerization to effectively couple the energy of ATP hydrolysis to the translocation of flagellar proteins.

Voltage-induced Infrared Absorption from a Spin-cast Thin Film of Ferroelectric Poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)).

Voltage-induced infrared spectra of annealed spin-cast thin films of ferroelectric poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) (molar ratio, 3:1) were measured in a stepwise cyclic external electric field. Most of the observed infrared bands originated from the ß ferroelectric crystalline phase. The voltage-induced spectral changes were decomposed into zeroth- (original), first-, and second-derivative spectra, and were attributed to the rotational motions of the polymer chains and the vibrational Stark effect. The values of the original spectral absorbance change ratios, ΔA/A, for the 849-cm-1 band (CF2 symmetric stretching, a1) and the 884-cm-1 band (CH2 rocking, b2) of the film exhibited double minimum and maximum peak hysteresis loops, respectively. The intensity of each band increased or decreased suddenly near a coercive field of ±0.6 MV/cm. These sudden intensity changes were attributed to the rotational inversion of the polymer chains that are associated with ferroelectricity.

Domain organization and function of Salmonella FliK, a flagellar hook-length control protein.

Salmonella hook-length control protein FliK, which consists of 405 amino acid residues, switches substrate specificity of the type III flagellar protein export apparatus from rod/ hook-type to filament-type by causing a conformational change in the cytoplasmic domain of FlhB (FlhB(C)) upon completion of the hook assembly. An N-terminal region of FliK contains an export signal, and a highly conserved C-terminal region consisting of amino acid residues 265-405 (FliK((265-405))) is directly involved in the switching of FlhB(C). Here, we have investigated the structural properties of FliK. Gel filtration chromatography, multi-angle light scattering and analytical ultracentrifugation showed that FliK is monomeric in solution and has an elongated shape. Limited proteolysis showed that FliK consists of two domains, the N-terminal (FliK(N)) and C-terminal domains (FliK(C)), and that the first 203 and the last 35 amino acid residues are partially unfolded and subjected to proteolysis. Both FliK(N) and FliK(C) are more globular than full-length FliK, suggesting that these domains are connected in tandem. Overproduced His-FliK((199-405)) failed to switch export specificity of the export apparatus. Affinity blotting revealed that FlhB(C) binds to FliK and FliK((1-147)), but not to FliK((265-405)). Based on these results, we propose that FliK(N) within the central channel of the hook-basal body during the export of FliK is the sensor and transmitter of hook completion information and that the binding interaction of FliK(C) to FlhB(C) is structurally regulated by FliK(N) so as to occur only when the hook has reached a preset length. The conformational flexibility of FliK(C) may play a role in interfering with switching at an inappropriate point of flagellar assembly.

Interactions between bacterial flagellar axial proteins in their monomeric state in solution.

The axial structure of the bacterial flagellum is composed of many different proteins, such as hook protein and flagellin, and each protein forms a short or long axial segment one after another in a well-defined order along the axis. Under physiological conditions, most of these proteins are stable in the monomeric state in solution, and spontaneous polymerization appears to be suppressed, as demonstrated clearly for flagellin, probably to avoid undesirable self-assembly in the cytoplasmic space. However, no systematic studies of the possible associations between monomeric axial proteins in solution have been carried out. We therefore studied self and cross-association between hook protein, flagellin and three hook-associated proteins, HAP1, HAP2 and HAP3, in all possible pairs, by gel-filtration and analytical centrifugation, and found interactions in the following two cases only. Flagellin facilitated HAP3 aggregation into beta-amyloid-like filaments, but without stable binding between the two. Addition of HAP3 to HAP2 resulted in disassembly of preformed HAP2 decamers and formation of stable HAP2-HAP3 heterodimers. HAP2 missing either of its disordered terminal regions did not form the heterodimer, whereas HAP3 missing either of its disordered terminal regions showed stable heterodimer formation. This polarity in the heterodimer interactions suggests that the interactions between HAP2 and HAP3 in solution are basically the same as those in the flagellar axial structure. We discuss these results in relation to the assembly mechanism of the flagellum.

Electric-Field-Induced Dynamics of Polymer Chains in a Ferroelectric Melt-Quenched Cold-Drawn Film of Nylon-11 Using Infrared Spectroscopy.

The changes in intensity of the infrared bands of a ferroelectric melt-quenched, cold-drawn film of nylon-11 were measured as a function of a cyclic external electric field of 1.4 MV/cm. The infrared bands assigned to the NH stretching, amide I, NH-vicinal, and CO-vicinal CH2 scissoring modes showed butterfly-shaped hysteresis loops that are characteristic of ferroelectrics; however, the intensity changes of the infrared bands assigned to the CH2 antisymmetric and symmetric stretching modes are small and showed no butterfly-shaped hysteresis loops. These results indicate that the amide groups are inverted, while the methylene groups are not inverted under the external electric field. We propose a new molecular mechanism that explains the ferroelectric properties of nylon-11. Only the amide groups in the antiparallel ß-sheet structure are inverted by the external electric field to form new hydrogen bonds; these two states form a nearly double-minimum potential.