Dimer Bands Research Articles

New infrared bands of nonpolar OCS dimer and experimental frequencies for two intermolecular modes

Spectra of the nonpolar carbonyl sulfide dimer in the region of the OCS ν(1) fundamental band were observed in a slit-jet supersonic expansion. The jet was probed using radiation from a tunable diode laser employed in a rapid-scan signal averaging mode. Six new bands were observed and analyzed, all of which originate from the dimer ground vibrational state. Three were vibrational fundamentals involving the ((18)OCS)(2) and (16)OCS-(18)OCS isotopologues. They enabled an estimate to be made of the frequency of the infrared-forbidden mode corresponding to in-phase vibration of the OCS monomers in the dimer, a value needed to obtain an intermolecular vibrational frequency from one of the observed combination bands. A relatively weak b-type dimer band centered at 2103.105 cm(-1) was assigned to the OCS 4ν(2) (l = 0) bending overtone. Combination bands were observed involving the geared bend and van der Waals stretch intermolecular modes. The resulting experimental frequencies of 37.5(20) cm(-1) for the bend and 42.9727(1) cm(-1) for the stretch are in good agreement with a recent high level theoretical calculation.

Antiparallel Dimers of the Small Multidrug Resistance Protein EmrE Are More Stable Than Parallel Dimers

The bacterial multidrug transporter EmrE is a dual-topology membrane protein and as such is able to insert into the membrane in two opposite orientations. The functional form of EmrE is a homodimer; however, the relative orientation of the subunits in the dimer is under debate. Using EmrE variants with fixed, opposite orientations in the membrane, we now show that, although the proteins are able to form parallel dimers, an antiparallel organization of the subunits in the dimer is preferred. Blue-native PAGE analyses of intact oligomers and disulfide cross-linking demonstrate that in membranes, the proteins form parallel dimers only if no oppositely orientated partner is present. Co-expression of oppositely orientated proteins almost exclusively yields antiparallel dimers. Finally, parallel dimers can be disrupted and converted into antiparallel dimers by heating of detergent-solubilized protein. Importantly, in vivo function is correlated clearly to the presence of antiparallel dimers. Our results suggest that an antiparallel arrangement of the subunits in the dimer is more stable than a parallel organization and likely corresponds to the functional form of the protein.

Abstract 410: 3-Dimensional Conformation of HDL ApoA-I Generated by ABCA1

This report describes the 3-dimensional conformation of nascent HDL apoA-I particles generated via ATP binding cassette transporter A1 (ABCA1). Three main types of particles were consistently obtained from; overexpressing HEK293 cells, bone marrow derived mouse macrophages and HepG2 cells. Particles were separated by FPLC and sized using gel electrophoresis and electron microscopy. Total lipid compositions were obtained from HPLC and mass spectrometry and protein conformation by chemical cross-linking and mass spectrometry (CCLMS). The first particle type corresponded to a lipid-rich nascent HDL (nHDL) between 13-10 nm in diameter, containing 3 molecules of apoA-I, with 45% of their total lipid as cholesterol, 35% as phosphatidylcholine and 20% as sphingomyelin with a total of ∼250 molecules of lipid per particle. The second type corresponded to 7-8 nm diameter particle containing 2 molecules of apoA-I and 26 total lipid molecules, while the third particle type was a monomer of apoA-I with 3 lipid molecules. Three-dimensional information was derived from the 13-10 nm nHDL particles by first crosslinking these purified particles and separating the monomeric, dimeric and trimeric apoA-I bands by SDS-PAGE. These CCL products were subjected to in-gel trypsin digest and LC-MS/MS. Reconstructed MS total-ion chromatograms were evaluated for CCL peptides with identity verified by MS/MS sequencing. Using several lysine-specific crosslinkers with arm lengths from 12Å to 36Å we compiled the distribution of CCLs. The same analysis was performed for the 7-8 nm nHDL that only yielded dimmer and monomer. Several intramolecular, interpeptide and intermolecular crosslinks were identified for each type category of nHDL. CCLs from the 7-8 nm nHDL suggested that the 2 apoA-I molecules assumed antiparallel counter-rotating helices. Tighter bending of the double belt was required to cover the 26 lipid molecules. Crosslinking of the 13-10 nm nHDL suggested that all three molecules were antiparallel to one another. The conformation that fit the CCLs was a belt with the middle apoA-I molecule antiparallel to the two outer apoA-I molecules. Overall, these data suggest that apoA-I adapts its conformation to encompass available lipid, but with larger amounts of lipid a belt conformation is preferred.

Vibrational spectra of monoisotopic SiH4 and GeH4 in low-temperature matrices

IR absorption spectra of monoisotopic 28SiH4 and 76GeH4 are studied in Ar and N2 matrices at 10 K. It is shown that the absorption spectra of silane and germane are similar in the regions of the stretching ν3 and bending ν4 vibrations. Four groups of bands can be separated out in the spectra of each molecule: (1) narrow bands characteristic of the matrix isolation studies, (2) broad bands, (3) diffuse absorption with a large value of the spectral moment M2* the intensity of which increases upon annealing, and (4) bands of dimers the intensity of which increases quadratically with concentration. The spectra of 28SiH4 and 76GeH4 in nitrogen matrices contain a triplet in the stretching region and a doublet in the bending region, which is explained by the change in the molecular symmetry from Td to C3V on passage from the gas phase to solid nitrogen.

Electron Transfer in Rhodobacter sphaeroides Reaction Centers Containing Zn-Bacteriochlorophylls: A Hole-Burning Study

Nonresonant and resonant transient, photochemical hole-burned (HB) spectra are presented for primary electron donor states of a novel bacterial reaction center (Zn-RC) of Rhodobacter sphaeroides, containing six Zn-bacteriochlorophylls (Zn-BChls). A "Zn-β-RC" in which the Zn-BChl in the bacteriopheophytin (BPhe)-binding site on the A side (H(A)) has the Zn penta-coordinated, was also studied. The fifth ligand comes from a histidine introduced by site-directed mutagenesis. Formation of the P(+)Q(A)(-) state was observed in both types of RC, although under identical experimental conditions a significantly deeper P(-) band (corresponding to the lower-energy, special pair, excitonic component) was revealed in the Zn-RC. Assuming a similar lifetime of the P(+)Q(A)(-) state, the quantum yield of P(+)Q(A)(-) formation decreased by ~60% in the Zn-β-RC (compared to the Zn-RC), as was seen in a comparison of analogous (Mg) BChl-containing wild type and β-RCs of Rb. sphaeroides [Kirmaier et al. Science1991, 251, 922]. However, the average (weakly frequency-dependent) low-temperature electron transfer (ET) rates of the Zn-RC and Zn-β-RC (measured from zero phonon holes in resonant transient HB spectra) were both ~1 ps and similar to a rate previously measured in the Rb. sphaeroides native RC [Johnson et al. J. Phys. Chem. 1989, 93, 5953]. Electron transfer rates observed in this work on the Zn-RC yielded a P870* decay rate in good agreement with recent room-temperature, time-domain data [Lin et al. Proc. Natl. Acad. Sci. 2009, 106, 8537]. A lack of correlation observed between the holes near 810 and 883 nm, accounting for electrochromically induced shifts of the Zn-BChl transitions in the B(A,B) and H(A,B) binding sites, produced by formation of the P(+)BHQ(A)(-) state, indicates that the 810 nm bleach does not correspond to the P(+) (upper excitonic component of the dimer) band and is mostly contributed to by a shift of the B(B) absorption band. ZPH-action spectra indicated inhomogeneous broadening (Γ(inh)) of ~110 cm(-1) (Zn-RC) and ~130 cm(-1) (Zn-β-RC). Experimentally determined Γ(inh) decreased the number of variables in theoretical fits of the absorption and frequency-dependent shapes of resonant HB spectra, leading to more reliable Huang-Rhys factors for both low-frequency phonons and a pseudolocalized phonon, ω(SP), often referred to as the special pair marker mode.

Cross-linking of serine racemase dimer by reactive oxygen species and reactive nitrogen species.

Serine racemase (SR) is the only identified enzyme in mammals responsible for isomerization of L-serine to D-serine, a coagonist at N-methyl-D-aspartate (NMDA) receptors in the forebrain. Our previous data showed that an apparent SR dimer resistant to sodium dodecyl sulfate and β-mercaptoethanol was elevated in microglial cells after proinflammatory activation. Because the activation of microglia is typically associated with an oxidative burst, oxidative cross-linking between SR subunits was speculated. In this study, an siRNA technique was employed to confirm the identity of this SR dimer band. The oxidative species potentially responsible for the cross-linking was investigated with recombinant SR protein. The data indicate that nitric oxide, peroxynitrite, and hydroxyl radical were the likely candidates, whereas superoxide and hydrogen peroxide per se failed to contribute. Furthermore, the mechanism of formation of SR dimer by peroxynitrite oxidation was studied by mass spectrometry. A disulfide bond between Cys₆ and Cys₁₁₃ was identified in 3-morpholinosydnonimine hydrochloride (SIN-1)-treated SR monomer and dimer. Activity assays indicated that SIN-1 treatment decreased SR activity, confirming our previous conclusion that noncovalent dimer is the most active form of SR. These findings suggest a compensatory feedback in which the consequences of neuroinflammation might dampen D-serine production to limit excitotoxic stimulation of NMDA receptors.

High‐molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Alzheimer’s disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer-soluble brain lysates were characterized in native form using blue native (BN)-PAGE and also in denatured form using SDS-PAGE followed by Western blot analysis. BN-PAGE analysis revealed a high-molecular weight smear (>1000 kD) of Aβ42-positive material in the AD brain, whereas low-molecular weight and monomeric Aβ species were not detected. SDS-PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ-positive material, thereby confirming the presence of high-molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ40- and Aβ42 preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN-PAGE. Further, BN-PAGE analysis exhibited a monomer band and less prominent low-molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS-PAGE showed large amounts of loMWAβ but no hiMWAβ40 and strikingly reduced levels of hiMWAβ42. These results indicate that hiMWAβ aggregates, particularly Aβ42 species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.

Digitonin and sodium dodecylsulfate-solubilized frog rho-dopsin: Behavior under native and denaturing polyacrylamide gel electrophoresis

Rhodopsin oligomerization and dissociation in vivo and under experimental conditions is an important topic both for a basic understanding of photoreceptor structure-function but also as a potential eye disease mechanism. In this study, to estimate a state rhodopsin after solubilization with mild and harsh detergents, we applied the native (blue native-PAGE, BN- PAGE) and denaturing electrophoresis (blue-urea- PAGE, BU-PAGE; blue-SDS-PAGE, BSDS-PAGE and SDS- PAGE). After blue BN-PAGE and BSDS- PAGE, rhodopsin and opsin, respectively, were presented in gels a major band of dimer with slight contents of higher oligomers without any traces of monomer, thus testifying in favor dimer-heteromeric state of frog rhodopsin in the photoreceptor membrane. Despite all oligomer bands gave positive staining with the rhodopsin-specific monoclonal antibodies (mAb), subsequent SDS-PAGE in combination with electroelution in denaturing conditions showed that stained bands are not homogenous and besides of opsin oli-gomers contain a small admixture of proteins with unknown function. Unfolding of opsin oligomers by solubilization in SDS, as compared with folded opsin in digitonin, induces their transition to a more compact conformation. It was manifested in a more rapid migration of opsin oligomers toward to anode. Cooling of digitonin/SDS mixed extracts at 4?C for 24 hours led to a partial reverse transition of unfolded opsin dimer to initial folded conformation, thus de- monstrating the entropic nature of this transition. Opsin monomer can be observed in the gels only after harsh dissociation of oligomers under BU-PAGE or SDS-PAGE. The electro elution of the individual opsin oligomers with denaturing buffer followed by SDS-PAGE resulted in dissociation of dimer to monomers. However, unexpectedly, the trimer was dissociated to a prevailing dimer and a small portion of monomer. The products dissociation of both opsin tetramer and pentamer are difficult to determine precisely, but they are neither monomer nor dimer. Dissociation data show that the degree of opsin oli- gomerization by unknown reasons affects the pattern of dissociation of its aggregates. Obtained in this paper data indicate a need for further detailed study the obscure mechanisms of aggregation-dissociation of rhodopsin.

High Resolution IR Spectroscopy of the C–H Stretch Bands of Benzene Monomer and Dimer in Helium Nanodroplets

Abstract The IR spectrum of benzene and benzene dimer embedded in superfluid helium droplets in the region of the C–H stretch vibration (3000–3110 cm-1) was observed. By analyzing the pressure dependence of the spectra it was possible to assign these spectra to benzene monomer and dimer. Based on a comparison of the observed position of the dimer bands to experimental bands recorded in the gas phase and to theoretical frequency predictions the authors assign these to the tilted T-shaped benzene dimer structure. None of the other two possible structures (PD or PTS) were found. The above assignment is firm. For the monomer, we find in helium nanodroplets a splitting of the ν 8+ν 19 combination band observed in the gas phase. We assign this to an additional Fermi resonance which is caused by the matrix shift in helium nanodroplets.

On the blue-shifting hydrogen bond in tribromoacetaldehyde dimers

The dimerisation of tribromoacetaldehyde in CCl4 and C6D12 solutions was investigated using Raman spectroscopy and different techniques of multivariate analysis. The ν(C–H) stretching vibration band in dimers was blue shifted by 14/15cm−1, whereas the ν(CO) dimer band was red-shifted by 7/8cm−1 in CCl4/C6D12 solutions versus their positions in monomers. The observed shifts were accompanied by a strong change in the bandwidth. The formation of dimers was confirmed by multivariate analysis of the Raman spectra of bromal recorded as a function of temperature. Structural and spectral information for (Br3CCHO)2 dimers were obtained from calculations at the B3LYP/6-311++G(3df,3pd) level.

Assembly of Therapeutic pRNA-siRNA Nanoparticles Using Bipartite Approach

The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles.

Observations of Host Guest Interactions Specific to Molecular Matrices: Water Monomers and Dimers in Hydrogen Matrices

Water monomers and dimers have been studied at low temperatures in matrices of solid p-H(2), o-D(2), n-H(2), and n-D(2) using infrared spectroscopy. Our data demonstrate interaction mechanisms between host matrix and guest molecules that are different from the ones observed in atomic noble gas matrices. Notably both guest/host rotational--rotational interaction and matrix induced modifications of the guest libration modes are observed. We also show that different types of interaction influence the relaxation times of some of guest modes. Water rotates freely in p-H(2) and o-D(2) but librates in n-H(2) and n-D(2). Rotational relaxation is faster in o-D(2) than in p-H(2) and faster in p-H(2) than in Ne. This is attributed to interactions between water rotation and matrix molecule rotation in p-H(2) and o-D(2). In n-H(2) and n-D(2), a strong water libration band is observed in the far-infrared, and strong water monomer vibration bands have libration satellites. Water dimer bands, close to matrix rotation bands, are perturbed by the matrix motions. The H-bonded isomer H(2)O--HOD rapidly converts to the D-bonded form H(2)O-DOH in p-H(2) and in o-D(2) but slowly in n-H(2) and n-D(2).

Architecture of a Full-length Retroviral Integrase Monomer and Dimer, Revealed by Small Angle X-ray Scattering and Chemical Cross-linking

We determined the size and shape of full-length avian sarcoma virus (ASV) integrase (IN) monomers and dimers in solution using small angle x-ray scattering. The low resolution data obtained establish constraints for the relative arrangements of the three component domains in both forms. Domain organization within the small angle x-ray envelopes was determined by combining available atomic resolution data for individual domains with results from cross-linking coupled with mass spectrometry. The full-length dimer architecture so revealed is unequivocally different from that proposed from x-ray crystallographic analyses of two-domain fragments, in which interactions between the catalytic core domains play a prominent role. Core-core interactions are detected only in cross-linked IN tetramers and are required for concerted integration. The solution dimer is stabilized by C-terminal domain (CTD-CTD) interactions and by interactions of the N-terminal domain in one subunit with the core and CTD in the second subunit. These results suggest a pathway for formation of functional IN-DNA complexes that has not previously been considered and possible strategies for preventing such assembly.

Intermolecular vibrational modes and orientational dynamics of cooperative hydrogen-bonding dimer of 7-azaindole in solution

We observed the low-frequency Raman-active intermolecular vibrational modes of 7-azaindole in CCl(4) by femtosecond Raman-induced Kerr effect spectroscopy. To understand the dynamical aspects and vibrational modes of 7-azaindole in the solution, the ultrafast dynamics of 1-benzofuran in CCl(4) was also examined as a reference and ab initio quantum chemistry calculations were performed for 7-azaindole and 1-benzofuran. The cooperative hydrogen-bonding vibrational bands of 7-azaindole dimer in CCl(4) appeared at 89 cm(-1) and 105 cm(-1) represent the overlap of stagger and wheeling modes and the intermolecular stretching mode, respectively. They are almost independent of the concentration in the solution. We further found from the low-frequency differential Kerr spectra of the solutions with neat CCl(4) that the intermolecular motion in the low frequency region below 20 cm(-1) was less active in the case of 7-azaindole/CCl(4) than in the case of 1-benzofuran/CCl(4). The slow orientational relaxation time in 7-azaindole/CCl(4) is ~3.5 times that in 1-benzofuran/CCl(4) because of the nature of the dimerization of 7-azaindole.

A synthetic gene increases TGFβ3 accumulation by 75‐fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule

Human transforming growth factor-β3 (TGFβ3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFβ3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFβ3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFβ3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFβ3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue-stained SDS-PAGE gels. TGFβ3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFβ3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13-kDa polypeptide into the TGFβ3 homodimer recognized by a conformation-dependent monoclonal antibody. The TGFβ3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFβ3. Functional equivalence was demonstrated by near-identical dose-response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFβ3.

Molecular characterization of the growth hormone in Chinese sturgeon and its expression during embryogenesis and early larval stages

Chinese sturgeon (Acipenser sinensis Gray) has a fast growth rate starting in early larval stage. Likely, the fast growth is attributed to the growth hormone (GH); an essential polypeptide required for growth and development of vertebrates. In this study, GH cDNA was cloned from a pituitary library. The AsGH cDNA consists approximately 954 bp in size including a 16 bp 5'-untranslated region and 296 bp 3'-untranslated region. The open reading frame (642 bp) encodes a 214 amino acids (aa), which represents the precursor composed of a 25 aa signal peptide followed by a 189 aa mature polypeptide. The polyclonal antibody was prepared from the in vitro expressed mature protein. Western blot analysis showed a constant GH expression of a monomeric form in the 13 embryo stages and in several larval stages. Interestingly, two dimeric bands of GH were detected since day 3 in the hatched larvae. Our findings suggest that the GH may play important physiological functions in embryonic and larval development of Chinese sturgeon.

Formation mechanisms of equilibrium component composition of molecular layers of polymethine dyes

We study the concentration dependences for the absorption spectra and component composition of molecular layers from three homologous series of symmetric polymethine dyes of different electron-donating ability of their terminal heterocyclic groups. We find that a change in the layer thickness leads to a change in the width and position of the spectrum due to a change in the number of absorption bands. The number of bands of monomers increases with increasing chain length and electron-donating ability. The concentration ratio of monomers and associated forms depends on the spatial orientation of molecules in the layer. The electron-donating ability of terminal groups affects the angle between the chromophores of molecules that form a dimer and the intensity ratio between the short- and long-wavelength absorption bands of dimers. We conclude that the effect of the thickness of the layer on its spectral parameters is determined by the degree of intramolecular electron asymmetry that arises as a result of the interaction of chemically symmetric molecules with charges of the substrate surface and upon intermolecular interaction. This asymmetry leads to changes in free energies of ground states of monomeric molecules and, as a consequence, to an increase in equilibrium concentrations of cis-stereoisomeric forms in the layer.

Cellulase Production by Pink Pigmented Facultative Methylotrophic Strains (PPFMs)

Pink pigmented facultative methylotrophs (PPFM) isolated from water samples of Cooum and Adyar rivers in Chennai and soil samples of forests located in various districts of Tamil Nadu, India were screened for cellulase production using carboxymethylcellulose agar (CMC agar) medium. The strains showed wide variations in the production of clearing zones around the colonies on CMC agar medium flooded with Congo red. CMCase and filter paper assays were used to quantitatively measure the cellulase activity of 13 PPFM strains. Among the strains, Methylobacterium gregans, MNW 60, MHW 109, MSF 34, and MSF 40 showed cellulolytic activity ranging from 0.73 to 1.16U mL(-1) with wide temperature (35-65°C) and pH (5 to 8) tolerance. SDS-PAGE analysis of the crude enzyme of PPFM strain MNW 60 exhibited several protein bands, and zymogram analysis revealed two dimeric cellulase bands with molecular mass of ~92 and 42kDa. Scanning electron microscopic studies revealed significant morphological differences between the cells grown in normal and CMC amended medium. The strain MNW 60 was identified as Methylobacterium sp. based on biochemical, physiological, and morphological analyses, and the methylotrophic nature was authenticated by the presence of mxaF gene, encoding methanol dehydrogenase as a key indicator enzyme of methylotrophs, with 99% similarity to Methylobacterium lusitanum. With the 16S ribosomal RNA sequence showing 97% similarity to M. lusitanum strain MP2, this can be proposed as a novel taxon of the genus Methylobacterium. The study forms the first detailed report on the extracellular cellulase production by pink pigmented Methylobacterium sp., and it is expected that this might be the basis for further studies on cellulase production by PPFMs to explore the molecular mechanism, strain improvement, and large-scale cellulase production for its application.

Characterization of the Oligomeric Structure of the Ca2+-activated Cl− Channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca(2+)-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl(-) secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca(2+), suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.