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.

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 Molecular Recognition of the Hybrid-Type G-Quadruplexes in Human Telomeres

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1578 ◽  
Author(s):  
Guanhui Wu ◽  
Luying Chen ◽  
Wenting Liu ◽  
Danzhou Yang
Keyword(s):  
Small Molecule ◽  
Solution Structure ◽  
Structural Information ◽  
Rational Drug Design ◽  
Hybrid Type ◽  
G4 Dna ◽  
Rational Drug ◽  
G Quadruplex ◽  
Molecule Recognition ◽  
Dna Secondary Structures

G-quadruplex (G4) DNA secondary structures formed in human telomeres have been shown to inhibit cancer-specific telomerase and alternative lengthening of telomere (ALT) pathways. Thus, human telomeric G-quadruplexes are considered attractive targets for anticancer drugs. Human telomeric G-quadruplexes are structurally polymorphic and predominantly form two hybrid-type G-quadruplexes, namely hybrid-1 and hybrid-2, under physiologically relevant solution conditions. To date, only a handful solution structures are available for drug complexes of human telomeric G-quadruplexes. In this review, we will describe two recent solution structural studies from our labs. We use NMR spectroscopy to elucidate the solution structure of a 1:1 complex between a small molecule epiberberine and the hybrid-2 telomeric G-quadruplex, and the structures of 1:1 and 4:2 complexes between a small molecule Pt-tripod and the hybrid-1 telomeric G-quadruplex. Structural information of small molecule complexes can provide important information for understanding small molecule recognition of human telomeric G-quadruplexes and for structure-based rational drug design targeting human telomeric G-quadruplexes.

scholarly journals Full Capacity of Recombinant Escherichia coliHeat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity

2000 ◽  
Vol 68 (7) ◽  
pp. 4064-4074 ◽  
Author(s):  
Isabelle Batisson ◽  
Maurice Der Vartanian ◽  
Brigitte Gaillard-Martinie ◽  
Michel Contrepois
Keyword(s):  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Biological Properties ◽  
Leader Peptide ◽  
Dependent Manner ◽  
Reporter Protein ◽  
E Coli ◽  
Heat Stable

ABSTRACT We have successfully used the major subunit ClpG ofEscherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2 or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

The Single Disulfide-Directed β-Hairpin Fold: Role of Disulfide Bond in Folding and Effect of an Additional Disulfide Bond on Stability

2020 ◽  
Vol 73 (4) ◽  
pp. 312
Author(s):  
Balasubramanyam Chittoor ◽  
Bankala Krishnarjuna ◽  
Rodrigo A. V. Morales ◽  
Raymond S. Norton
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Conformational Stability ◽  
Disulfide Bridge ◽  
Solution Nmr ◽  
Peptide Epitope ◽  
The Core ◽  
Native Fold

Disulfide bonds play a key role in the oxidative folding, conformational stability, and functional activity of many peptides. A few disulfide-rich peptides with privileged architecture such as the inhibitor cystine knot motif have garnered attention as templates in drug design. The single disulfide-directed β-hairpin (SDH), a novel fold identified more recently in contryphan-Vc1, has been shown to possess remarkable thermal, conformational, and chemical stability and can accept a short bioactive epitope without compromising the core structure of the peptide. In this study, we demonstrated that the single disulfide bond is critical in maintaining the native fold by replacing both cysteine residues with serine. We also designed an analogue with an additional, non-native disulfide bridge by replacing Gln1 and Tyr9 with Cys. Contryphan-Vc11–22[Q1C, Y9C] was synthesised utilising orthogonal cysteine protection and its solution structure determined using solution NMR spectroscopy. This analogue maintained the overall fold of native contryphan-Vc1. Previous studies had shown that the β-hairpin core of contryphan-Vc1 was resistant to proteolysis by trypsin and α-chymotrypsin but susceptible to cleavage by pepsin. Contryphan-Vc11–22[Q1C, Y9C] proved to be completely resistant to pepsin, thus confirming our design strategy. These results highlight the role of the disulfide bond in maintaining the SDH fold and provide a basis for the design of more stable analogues for peptide epitope grafting.

scholarly journals Long-Range RNA Structural Information via a Paramagnetically Tagged Reporter Protein

2019 ◽  
Vol 141 (4) ◽  
pp. 1430-1434 ◽  
Author(s):  
Madeleine Strickland ◽  
Jonathan Catazaro ◽  
Rohith Rajasekaran ◽  
Marie-Paule Strub ◽  
Colin O’Hern ◽  
...  
Keyword(s):  
Long Range ◽  
Structural Information ◽  
Reporter Protein

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 The susceptibility of disulfide bonds towards radiation damage may be explained by S...O interactions

IUCrJ ◽  
2020 ◽  
Vol 7 (5) ◽  
pp. 825-834
Author(s):  
Rajasri Bhattacharyya ◽  
Jesmita Dhar ◽  
Shubhra Ghosh Dastidar ◽  
Pinak Chakrabarti ◽  
Manfred S. Weiss
Keyword(s):  
Electron Transfer ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Metal Ion ◽  
Structural Information ◽  
Close Contact ◽  
Bond Cleavage ◽  
Protein Structures ◽  
Protein Crystal ◽  
X Ray

Radiation-induced damage to protein crystals during X-ray diffraction data collection is a major impediment to obtaining accurate structural information on macromolecules. Some of the specific impairments that are inflicted upon highly brilliant X-ray irradiation are metal-ion reduction, disulfide-bond cleavage and a loss of the integrity of the carboxyl groups of acidic residues. With respect to disulfide-bond reduction, previous results have indicated that not all disulfide bridges are equally susceptible to damage. A careful analysis of the chemical environment of disulfide bonds in the structures of elastase, lysozyme, acetylcholinesterase and other proteins suggests that S—S bonds which engage in a close contact with a carbonyl O atom along the extension of the S—S bond vector are more susceptible to reduction than the others. Such an arrangement predisposes electron transfer to occur from the O atom to the disulfide bond, leading to its reduction. The interaction between a nucleophile and an electrophile, akin to hydrogen bonding, stabilizes protein structures, but it also provides a pathway of electron transfer to the S—S bond, leading to its reduction during exposure of the protein crystal to an intense X-ray beam. An otherwise stabilizing interaction can thus be the cause of destabilization under the condition of radiation exposure.

scholarly journals Structure-Based Functional Analysis of a Hormone Belonging to an Ecdysozoan Peptide Superfamily: Revelation of a Common Molecular Architecture and Residues Possibly for Receptor Interaction

2021 ◽  
Vol 22 (20) ◽  
pp. 11142
Author(s):  
Yun-Ru Chen ◽  
Nai-Wan Hsiao ◽  
Yi-Zong Lee ◽  
Shiau-Shan Huang ◽  
Chih-Chun Chang ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Receptor Binding ◽  
Binding Sites ◽  
Disulfide Bonds ◽  
Solution Structure ◽  
Receptor Interaction ◽  
Resonance Spectroscopy ◽  
Molecular Architecture ◽  
Major Factors

A neuropeptide (Sco-CHH-L), belonging to the crustacean hyperglycemic hormone (CHH) superfamily and preferentially expressed in the pericardial organs (POs) of the mud crab Scylla olivacea, was functionally and structurally studied. Its expression levels were significantly higher than the alternative splice form (Sco-CHH) in the POs, and increased significantly after the animals were subjected to a hypo-osmotic stress. Sco-CHH-L, but not Sco-CHH, significantly stimulated in vitro the Na+, K+-ATPase activity in the posterior (6th) gills. Furthermore, the solution structure of Sco-CHH-L was resolved using nuclear magnetic resonance spectroscopy, revealing that it has an N-terminal tail, three α-helices (α2, Gly9−Asn28; α3, His34−Gly38; and α5, Glu62−Arg72), and a π-helix (π4, Cys43−Tyr54), and is structurally constrained by a pattern of disulfide bonds (Cys7–Cys43, Cys23–Cys39, and Cys26–Cys52), which is characteristic of the CHH superfamily-peptides. Sco-CHH-L is topologically most similar to the molt-inhibiting hormone from the Kuruma prawn Marsupenaeus japonicus with a backbone root-mean-square-deviation of 3.12 Å. Ten residues of Sco-CHH-L were chosen for alanine-substitution, and the resulting mutants were functionally tested using the gill Na+, K+-ATPase activity assay, showing that the functionally important residues (I2, F3, E45, D69, I71, and G73) are located at either end of the sequence, which are sterically close to each other and presumably constitute the receptor binding sites. Sco-CHH-L was compared with other members of the superfamily, revealing a folding pattern, which is suggested to be common for the crustacean members of the superfamily, with the properties of the residues constituting the presumed receptor binding sites being the major factors dictating the ligand–receptor binding specificity.

Mitochondrial targeting of yeast apoiso-1-cytochrome c is mediated through functionally independent structural domains

1990 ◽  
Vol 10 (11) ◽  
pp. 5763-5771
Author(s):  
S H Nye ◽  
R C Scarpulla
Keyword(s):  
Cytochrome C ◽  
Mitochondrial Membrane ◽  
Structural Information ◽  
Alpha Helix ◽  
Initial Step ◽  
Intermembrane Space ◽  
Mitochondrial Targeting ◽  
Structural Determinants ◽  
Amino Terminal

An iso-1-cytochrome c-chloramphenicol acetyltransferase fusion protein (iso-1/CAT) was expressed in Saccharomyces cerevisiae and used to delineate two stages in the cytochrome c import pathway in vivo (S. H. Nye and R. C. Scarpulla, Mol. Cell. Biol. 10:5753-5762, 1990 [this issue]). Fusion proteins with the CAT reporter domain in its native conformation were arrested at the initial stage of mitochondrial membrane recognition and insertion. In contrast, those with a deletional disruption of the CAT moiety were relieved of this block and allowed to translocate to the intermembrane space, where they functioned in respiratory electron transfer. In the present study, iso-1/CAT was used to map structural determinants in apoiso-1-cytochrome c involved in the initial step of targeting to the mitochondrial membrane. Carboxy-terminal deletions revealed that one of these determinants consisted of the amino-terminal 68 residues. Deletion mutations either within or at the ends of this determinant destroyed mitochondrial targeting activity, suggesting that functionally important information spans the length of this fragment. Disruption of an alpha-helix near the amino terminus by a helix-breaking proline substitution for leucine 14 also eliminated the targeting activity of the 1 to 68 determinant, suggesting a contribution from this structure. A second, functionally independent targeting determinant was found in the carboxy half of the apoprotein between residues 68 and 85. This determinant coincided with a stretch of 11 residues that are invariant in nearly 100 eucaryotic cytochromes c. Therefore, in lieu of an amino-terminal presequence, apocytochrome c has redundant structural information located in both the amino and carboxy halves of the molecule that can function independently to specify mitochondrial targeting and membrane insertion in vivo.

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