Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag

2011 ◽  
Vol 1814 (12) ◽  
pp. 1775-1778 ◽  
Author(s):  
Tharangani Rathnayaka ◽  
Minako Tawa ◽  
Takashi Nakamura ◽  
Shihori Sohya ◽  
Kunihiro Kuwajima ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Gaussia Luciferase

scholarly journals Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

2020 ◽  
Author(s):  
Nan Wu ◽  
Naohiro Kobayashi ◽  
Kengo Tsuda ◽  
Satoru Unzai ◽  
Tomonori Saotome ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Solution Structure ◽  
Heteronuclear Nmr ◽  
Structural Information ◽  
Alpha Helix ◽  
Reporter Protein ◽  
Gaussia Luciferase ◽  
Hydrophobic Cavity ◽  
Binding Cavity ◽  
Heteronuclear Multidimensional Nmr

AbstractGaussia luciferase (GLuc) is the smallest luciferase (18.2kDa; 168 residues) reported so far and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a solubility tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36-81, 96-145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 ű 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

scholarly journals Solution structure of Gaussia Luciferase with five disulfide bonds and identification of a putative coelenterazine binding cavity by heteronuclear NMR

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nan Wu ◽  
Naohiro Kobayashi ◽  
Kengo Tsuda ◽  
Satoru Unzai ◽  
Tomonori Saotome ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Solution Structure ◽  
Heteronuclear Nmr ◽  
Structural Information ◽  
Alpha Helix ◽  
Reporter Protein ◽  
Gaussia Luciferase ◽  
Hydrophobic Cavity ◽  
Binding Cavity ◽  
Heteronuclear Multidimensional Nmr

AbstractGaussia luciferase (GLuc) is a small luciferase (18.2 kDa; 168 residues) and is thus attracting much attention as a reporter protein, but the lack of structural information is hampering further application. Here, we report the first solution structure of a fully active, recombinant GLuc determined by heteronuclear multidimensional NMR. We obtained a natively folded GLuc by bacterial expression and efficient refolding using a Solubility Enhancement Petide (SEP) tag. Almost perfect assignments of GLuc’s 1H, 13C and 15N backbone signals were obtained. GLuc structure was determined using CYANA, which automatically identified over 2500 NOEs of which > 570 were long-range. GLuc is an all-alpha-helix protein made of nine helices. The region spanning residues 10–18, 36–81, 96–145 and containing eight out of the nine helices was determined with a Cα-atom RMSD of 1.39 Å ± 0.39 Å. The structure of GLuc is novel and unique. Two homologous sequential repeats form two anti-parallel bundles made by 4 helices and tied together by three disulfide bonds. The N-terminal helix 1 is grabbed by these 4 helices. Further, we found a hydrophobic cavity where several residues responsible for bioluminescence were identified in previous mutational studies, and we thus hypothesize that this is a catalytic cavity, where the hydrophobic coelenterazine binds and the bioluminescence reaction takes place.

Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

Specific Cross-reaction of IgG Anti-phospholipid Antibody with Platelet Glycoprotein IIIa

1996 ◽  
Vol 75 (01) ◽  
pp. 168-174 ◽  
Author(s):  
Shigeru Tokita ◽  
Morio Arai ◽  
Naomasa Yamamoto ◽  
Yasuhiro Katagiri ◽  
Kenjiro Tanoue ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Heparan Sulfate ◽  
Disulfide Bonds ◽  
Dextran Sulfate ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent Fashion ◽  
Glycoprotein Iiia

SummaryTo study the pathological functions of anti-phospholipid (anti-PL) antibodies, we have analyzed their effect on platelet function. We identified an IgG anti-PL mAb, designated PSG3, which cross-reacted specifically with glycoprotein (GP) IIIa in human platelets and inhibited platelet aggregation. PSG3 bound also to certain polyanionic substances, such as double-stranded DNA, heparan sulfate, dextran sulfate and acetylated-LDL, but not to other polyanionic substances. The binding of PSG3 to GPIIIa was completely inhibited by heparan sulfate and dextran sulfate, indicating that PSG3 recognizes a particular array of negative charges expressed on both GPIIIa and the specified polyanionic substances. Since neither neuraminidase- nor endoglycopeptidase F-treatment of GPIIIa had any significant effect on the binding of PSG3, this array must be located within the amino acid sequence of GPIIIa but not in the carbohydrate moiety. Reduction of the disulfide bonds in GPIIIa greatly reduced its reactivity, suggesting that the negative charges in the epitope are arranged in a particular conformation. PSG3 inhibited platelet aggregation induced by either ADP or collagen, it also inhibited fibrinogen binding to activated platelets in a dose-dependent fashion. PSG3, however, did not inhibit the binding of GRGDSP peptide to activated platelets. These results suggest that the PSG3 epitope on GPIIIa contains a particular array of negative charges, and possibly affects the fibrinogen binding to GPIIb/IIIa complex necessary for platelet aggregation.

Peptides from peptic digest of bovine DIP-tripsin and determination of disulfide bonds

1968 ◽  
Vol 33 (2) ◽  
pp. 441-461 ◽  
Author(s):  
V. Holeyšovský ◽  
B. Mesrob ◽  
V. Tomášek ◽  
O. Mikeš ◽  
F. Šorm
Keyword(s):  
Disulfide Bonds

scholarly journals pH-activated, mitochondria-targeted, and redox-responsive delivery of paclitaxel nanomicelles to overcome drug resistance and suppress metastasis in lung cancer

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
He Wang ◽  
Wenwen Shi ◽  
Danning Zeng ◽  
Qiudi Huang ◽  
Jiacui Xie ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Drug Resistance ◽  
Disulfide Bonds ◽  
Cancer Metastasis ◽  
Mitochondrial Outer Membrane ◽  
Drug Resistant ◽  
Redox Responsive ◽  
Dimethylmaleic Anhydride ◽  
Extended Circulation ◽  
Mitochondria Targeting

Abstract Background Mitochondria play a role in the occurrence, development, drug resistance, metastasis, and other functions of cancer and thus are a drug target. An acid-activated mitochondria-targeting drug nanocarrier with redox-responsive function was constructed in the present study. However, whether this vector can precisely delivery paclitaxel (PTX) to enhance therapeutic efficacy in drug-resistant lung cancer is unknown. Results Acid-cleavable dimethylmaleic anhydride (DA) was used to modify pluronic P85-conjugated mitochondria-targeting triphenylphosphonium (TPP) using disulfide bonds as intermediate linkers (DA-P85-SS-TPP and DA-P-SS-T). The constructed nanocarriers demonstrated enhanced cellular uptake and selective mitochondrial targeting at extracellular pH characteristic for a tumor (6.5) and were characterized by extended circulation in the blood. TPP promoted the targeting of the DA-P-SS-T/PTX nanomicelles to the mitochondrial outer membrane to decrease the membrane potential and ATP level, resulting in inhibition of P-glycoprotein and suppression of drug resistance and cancer metastasis. PTX was also rapidly released in the presence of high glutathione (GSH) levels and directly diffused into the mitochondria, resulting in apoptosis of drug-resistant lung cancer cells. Conclusions These promising results indicated that acid-activated mitochondria-targeting and redox-responsive nanomicelles potentially represent a significant advancement in cancer treatment. Graphic Abstarct

scholarly journals General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shay Laps ◽  
Fatima Atamleh ◽  
Guy Kamnesky ◽  
Hao Sun ◽  
Ashraf Brik
Keyword(s):  
Drug Discovery ◽  
Disulfide Bonds ◽  
Unsolved Problem ◽  
De Novo ◽  
Therapeutic Potential ◽  
Selective Activation ◽  
One Pot ◽  
Synthetic Strategy ◽  
The One ◽  
Game Changer

AbstractDespite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.

scholarly journals Kinetical Study, Thermo-Mechanical Characteristics and Recyclability of Epoxidized Camelina Oil Cured with Antagonist Structure (Aliphatic/Aromatic) or Functionality (Acid/Amine) Hardeners

Polymers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 2503
Author(s):  
Chiara Di Mauro ◽  
Aratz Genua ◽  
Alice Mija
Keyword(s):  
Disulfide Bonds ◽  
Mechanical Characteristics ◽  
Dicarboxylic Acids ◽  
Thermomechanical Properties ◽  
Chemical Recycling ◽  
Camelina Oil ◽  
Ft Ir ◽  
Tan Δ ◽  
The Individual ◽  
First Time

In an attempt to prepare sustainable epoxy thermosets, this study introduces for the first time the idea to use antagonist structures (aromatic/aliphatic) or functionalities (acid/amine) as hardeners to produce reprocessable resins based on epoxidized camelina oil (ECMO). Two kinds of mixtures were tested: one combines aromatic/aliphatic dicarboxylic acids: 2,2′-dithiodibenzoic acid (DTBA) and 3,3′-dithiodipropionic acid (DTDA); another is the combination of two aromatic structures with acid/amine functionality: DTBA and 4-aminophenyl disulfide (4-AFD). DSC and FT-IR analyses were used as methods to analyze the curing reaction of ECMO with the hardeners. It was found that the thermosets obtained with the dual crosslinked mechanism needed reduced curing temperatures and reprocessing protocols compared to the individual crosslinked thermosets. Thanks to the contribution of disulfide bonds in the network topology, the obtained thermosets showed recycling ability. The final thermomechanical properties of the virgin and mechanical reprocessed materials were analyzed by DMA and TGA. The obtained thermosets range from elastomeric to rigid materials. As an example, the ECMO/DTBA704-AFD30 virgin or reprocessed thermosets have tan δ values reaching 82–83 °C. The study also investigates the chemical recycling and the solvent resistance of these vitrimer-like materials.

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