scholarly journals Bombyx mori Midgut Membrane Protein P252, Which Binds to Bacillus thuringiensis Cry1A, Is a Chlorophyllide-Binding Protein, and the Resulting Complex Has Antimicrobial Activity

2008 ◽  
Vol 74 (5) ◽  
pp. 1324-1331 ◽  
Author(s):  
Ganesh N. Pandian ◽  
Toshiki Ishikawa ◽  
Makoto Togashi ◽  
Yasuyuki Shitomi ◽  
Kohsuke Haginoya ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Thuringiensis ◽  
Bombyx Mori ◽  
Conformational Changes ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Binding Characteristics ◽  
Α Helix ◽  
Negligible Contribution

ABSTRACT The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 μM is shown to bind with about 50 μM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37°C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a β-structure (39.8% ± 2.2%, based on 5 samples) with negligible contribution of α-helix structure. When bound to Chlide, the β-structure content in the complex is reduced to 21.6% ± 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 μM, and 21.6 μM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.

Physico-Chemical Studies of Isolated Chromatin Compared with in situ Chromatin after Partial Hepatectomy in the Rat

1979 ◽  
Vol 34 (5-6) ◽  
pp. 442-448 ◽  
Author(s):  
Paul Miller ◽  
Walfried A. Linden ◽  
Claudio Nicolini
Keyword(s):  
Partial Hepatectomy ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Cultured Cells ◽  
Fluorescence Emission ◽  
Chromatin Organization ◽  
Chromatin Conformation ◽  
Functional Changes

Abstract Chromatin was isolated from rat liver cells at 0, 3, 5, 11, 18 and 24 h following partial hepatectomy. Consistent with findings in cultured cells stimulated to proliferate, there was an increase in chromatin molar ellipticity measured at 276 nm, and a decrease in thermal stability 3 to 8 h after surgery. These events occured prior to the onset of DNA synthesis. These early changes be­ tween non-proliferating (G0) and proliferating (G1) cells, as well as later chromatin conformational changes observed at S and G2 phases, mimic changes in template activity.Results with sheared and unsheared chromatin (both with in vitro and in vivo systems) prove that structural and functional changes can be caused by even the slightest shearing during chromatin preparation, suggesting the loss of native chromatin organization. To eliminate this problem, experiments were also conducted using chromatin in situ. A flow cytometer (FCM) was used to study unfixed liver cell suspensions stained with ethidium bromide (EB). Fluorescence was mea­ sured in the green spectral range after addition of increasing amounts of EB. Experimental evidence is provided that the same alteration in chromatin conformation can be best detected using low molar ratios of EB per unit DNA due to greater fluorescence emission in G1 respect to G0 cells.These correlated studies demonstrate that the same changes controlling chromatin organization in situ are detected also in the tertiary-quaternary structure of “isolated” chromatin. These changes in chromatin conformation are macromolecular events related to cell proliferation both at the G0 -G1 and G1 -S transitions.

scholarly journals Interaction of BAG1 and Hsp70 Mediates Neuroprotectivity and Increases Chaperone Activity

2005 ◽  
Vol 25 (9) ◽  
pp. 3715-3725 ◽  
Author(s):  
Jan Liman ◽  
Sundar Ganesan ◽  
Christoph P. Dohm ◽  
Stan Krajewski ◽  
John C. Reed ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Heat Shock Protein 70 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Full Length ◽  
Wild Type ◽  
Binding Characteristics ◽  
In Cells

ABSTRACT It was recently shown that Bcl-2-associated athanogene 1 (BAG1) is a potent neuroprotectant as well as a marker of neuronal differentiation. Since there appears to exist an equilibrium within the cell between BAG1 binding to heat shock protein 70 (Hsp70) and BAG1 binding to Raf-1 kinase, we hypothesized that changing BAG1 binding characteristics might significantly alter BAG1 function. To this end, we compared rat CSM14.1 cells and human SHSY-5Y cells stably overexpressing full-length BAG1 or a deletion mutant (BAGΔC) no longer capable of binding to Hsp70. Using a novel yellow fluorescent protein-based foldase biosensor, we demonstrated an upregulation of chaperone in situ activity in cells overexpressing full-length BAG1 but not in cells overexpressing BAGΔC compared to wild-type cells. Interestingly, in contrast to the nuclear and cytosolic localizations of full-length BAG1, BAGΔC was expressed exclusively in the cytosol. Furthermore, cells expressing BAGΔC were no longer protected against cell death. However, they still showed accelerated neuronal differentiation. Together, these results suggest that BAG1-induced activation of Hsp70 is important for neuroprotectivity, while BAG1-dependent modulation of neuronal differentiation in vitro is not.

Binding to lipid membrane induces conformational changes in RPE65: implications for its isomerohydrolase activity

2011 ◽  
Vol 436 (3) ◽  
pp. 591-597 ◽  
Author(s):  
Olga Nikolaeva ◽  
Gennadiy Moiseyev ◽  
Karla K. Rodgers ◽  
Jian-xing Ma
Keyword(s):  
Fluorescence Quenching ◽  
Conformational Changes ◽  
Lipid Membrane ◽  
Membrane Binding ◽  
Tryptophan Fluorescence ◽  
Cd Spectroscopy ◽  
Visual Cycle ◽  
Α Helix ◽  
Structural Levels

The visual cycle is a multi-step pathway to recycle 11-cis retinal, the chromophore for both rod and cone visual pigments. The isomerohydrolase RPE65, a membrane-associated enzyme, converts atRE (all-trans-retinyl ester) to 11-cis-retinol, a key step in the visual cycle. Previously, it has been shown that membrane association of RPE65 is essential for its catalytic activity. Using purified recombinant chicken RPE65 and an in vitro liposome-based floatation assay, we present evidence that the RPE65 membrane-binding affinity was significantly facilitated by incorporation of atRE, the substrate of RPE65, into liposomal membrane. Using tryptophan emission fluorescence quenching and CD spectroscopy, we showed that, upon membrane binding, RPE65 undergoes conformational changes at both the tertiary and secondary structural levels. Specifically, tryptophan fluorescence quenching showed that the tertiary RPE65 structure became more open towards the hydrophilic environment upon its association with the membrane. Simultaneously, a decrease in the α-helix content of RPE65 was revealed upon binding with the lipid membrane containing atRE. These results demonstrated that RPE65's functional activity depends on its conformational changes caused by its association with the membrane.

scholarly journals A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles

2020 ◽  
Vol 295 (8) ◽  
pp. 2285-2298
Author(s):  
Samantha K. Sarkar ◽  
Alexander C. Y. Foo ◽  
Angela Matyas ◽  
Ikhuosho Asikhia ◽  
Tanja Kosenko ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Loss Of Function ◽  
Ldl Particles ◽  
Ldl Binding ◽  
Α Helix

Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein (LDL) receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9's prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish PCSK9's ability to bind LDL reported here supports the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

scholarly journals Pegylation of Antimicrobial Peptides Maintains the Active Peptide Conformation, Model Membrane Interactions, and Antimicrobial Activity while Improving Lung Tissue Biocompatibility following Airway Delivery

2012 ◽  
Vol 56 (6) ◽  
pp. 3298-3308 ◽  
Author(s):  
Christopher J. Morris ◽  
Konrad Beck ◽  
Marc A. Fox ◽  
David Ulaeto ◽  
Graeme C. Clark ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Lung Tissue ◽  
Conformational Changes ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Membrane Integrity ◽  
Pulmonary Infections ◽  
Cecropin A

ABSTRACTAntimicrobial peptides (AMPs) have therapeutic potential, particularly for localized infections such as those of the lung. Here we show that airway administration of a pegylated AMP minimizes lung tissue toxicity while nevertheless maintaining antimicrobial activity. CaLL, a potent synthetic AMP (KWKLFKKIFKRIVQRIKDFLR) comprising fragments of LL-37 and cecropin A peptides, was N-terminally pegylated (PEG-CaLL). PEG-CaLL derivatives retained significant antimicrobial activity (50% inhibitory concentrations [IC50s] 2- to 3-fold higher than those of CaLL) against bacterial lung pathogens even in the presence of lung lining fluid. Circular dichroism and fluorescence spectroscopy confirmed that conformational changes associated with the binding of CaLL to model microbial membranes were not disrupted by pegylation. Pegylation of CaLL reduced AMP-elicited cell toxicity as measured usingin vitrolung epithelial primary cell cultures. Further, in a fully intactex vivoisolated perfused rat lung (IPRL) model, airway-administered PEG-CaLL did not result in disruption of the pulmonary epithelial barrier, whereas CaLL caused an immediate loss of membrane integrity leading to pulmonary edema. All AMPs (CaLL, PEG-CaLL, LL-37, cecropin A) delivered to the lung by airway administration showed limited (<3%) pulmonary absorption in the IPRL with extensive AMP accumulation in lung tissue itself, a characteristic anticipated to be beneficial for the treatment of pulmonary infections. We conclude that pegylation may present a means of improving the lung biocompatibility of AMPs designed for the treatment of pulmonary infections.

scholarly journals Kinesin-binding protein remodels the kinesin motor to prevent microtubule-binding

2021 ◽  
Author(s):  
April L Solon ◽  
Zhenyu Tan ◽  
Katherine L Schutt ◽  
Lauren Jepsen ◽  
Sarah E Haynes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Protein ◽  
Structural Element ◽  
Kinesin Motor ◽  
Microtubule Binding ◽  
Structural Mechanism ◽  
Binding Interface ◽  
Α Helix ◽  
Motor Head

Kinesins are tightly regulated in space and time to control their activation in the absence of cargo-binding. Kinesin-binding protein (KIFBP) was recently discovered to bind the catalytic motor heads of 8 of the 45 known kinesin superfamily members and inhibit binding to microtubules. In humans, mutation of KIFBP gives rise to Goldberg-Shprintzen syndrome (GOSHS), but the kinesin(s) that is misregulated to produce clinical features of the disease is not known. Understanding the structural mechanism by which KIFBP selects its kinesin binding partners will be key to unlocking this knowledge. Using a combination of cryo-electron microscopy and crosslinking mass spectrometry, we determined structures of KIFBP alone and in complex with two mitotic kinesins, revealing regions of KIFBP that participate in complex formation. KIFBP adopts an alpha-helical solenoid structure composed of TPR repeats. We find that KIFBP uses a 2-pronged mechanism to remodel kinesin motors and block microtubule-binding. First, KIFBP engages the microtubule-binding interface and sterically blocks interaction with microtubules. Second, KIFBP induces allosteric conformational changes to the kinesin motor head that displace a key structural element in the kinesin motor head (α-helix 4) required for microtubule binding. We identified two regions of KIFBP necessary for in vitro kinesin-binding as well as cellular regulation during mitosis. Taken together, this work establishes the mechanism of kinesin inhibition by KIFBP and provides the first example of motor domain remodeling as a means to abrogate kinesin activity.

Identification of In-Vitro Red Fluorescent Protein with Antipathogenic Activity from the Midgut of the Silkworm (Bombyx Mori L.)

2018 ◽  
Vol 25 (3) ◽  
pp. 302-313 ◽  
Author(s):  
Manjunatha ◽  
Anitha Peter ◽  
Mahantesha B.N. Naika ◽  
P. Niranjana ◽  
P. Shamprasad
Keyword(s):  
Bombyx Mori ◽  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Bombyx Mori L ◽  
Silkworm Bombyx Mori

scholarly journals An Engineered Chymotrypsin/Cathepsin G Site in Domain I Renders Bacillus thuringiensis Cry3A Active against Western Corn Rootworm Larvae

2007 ◽  
Vol 74 (2) ◽  
pp. 367-374 ◽  
Author(s):  
Frederick S. Walters ◽  
Cheryl M. Stacy ◽  
Mi Kyong Lee ◽  
Narendra Palekar ◽  
Jeng S. Chen
Keyword(s):  
Bacillus Thuringiensis ◽  
Specific Binding ◽  
The United States ◽  
Western Corn Rootworm ◽  
Cathepsin G ◽  
Corn Rootworm ◽  
Α Helix ◽  
Domain I

ABSTRACTThe western corn rootworm remains one of the most important pests of corn in the United States despite the use of many pest management tools. Cry3A, the first coleopteran-activeBacillus thuringiensistoxin isolated, has not been useful for control of the corn rootworm pest complex. Modification of Cry3A so that it contained a chymotrypsin/cathepsin G protease recognition site in the loop between α-helix 3 and α-helix 4 of domain I, however, resulted in consistent activity of the toxin (“mCry3A”) against neonate western corn rootworm. In vitro chymotrypsin digests showed that there was a substantial difference between the enzyme sensitivity of mCry3A and the enzyme sensitivity of Cry3A, with mCry3A rapidly converted from a 67-kDa form to a ∼55-kDa form. The introduced protease site was also recognized in vivo, where the ∼55-kDa form of mCry3A toxin was rapidly generated and associated with the membrane fraction. After a point mutation in mcry3Athat resulted in the elimination of the native domain I chymotrypsin site (C terminal to the introduced chymotrypsin/cathepsin G protease site of mCry3A), the in vitro and in vivo digestion patterns remained the same, demonstrating that the introduced site was required for the enhanced activity. Also, 55-kDa mCry3A generated by cleavage with chymotrypsin exhibited specific binding to western corn rootworm brush border membrane, whereas untreated 67-kDa mCry3A did not. These data indicate that the mCry3A toxicity for corn rootworm larvae was due to the introduction of a chymotrypsin/cathepsin G site, which enhanced cleavage and subsequent binding of the activated toxin to midgut cells.

scholarly journals A transient amphipathic helix in PCSK9’s prodomain facilitates low-density lipoprotein binding

2019 ◽  
Author(s):  
Samantha K. Sarkar ◽  
Alexander C.Y. Foo ◽  
Angela Matyas ◽  
Tanja Kosenko ◽  
Natalie K. Goto ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Helical Structure ◽  
Low Density ◽  
Loss Of Function ◽  
Ldl Particles ◽  
Ldl Binding ◽  
Α Helix

SUMMARYProprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30-40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9’s prodomain. Here we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N-terminus adopted helical structure in a membrane-mimetic environment; this effect was greatly enhanced by an R46L substitution representing an athero-protective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested the transient α-helix aligns multiple polar residues to interact with positive-charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, F515L) inhibited LDL binding, which was abolished for the R496W variant. These studies inform on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, we report the initial identification of FH-associated mutations that diminish the ability of PCSK9 to bind LDL, supporting that LDL association in the circulation inhibits PCSK9 activity.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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