Release of the Neocarzinostatin Chromophore from the Holoprotein Does Not Require Major Conformational Change of the Tertiary and Secondary Structures Induced by Trifluoroethanol

Christopher, G.; Sudhahar, P.; Balamurugan, Krishnaswamy; Chin, Daeje

doi:10.1074/jbc.m006837200

Cited by 18 publications

(1 citation statement)

References 44 publications

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“…All spectra were corrected for the respective buffer blanks and data are expressed as mean residue ellipticity [q]. [54] Thermal-induced protein unfolding experiments were conducted in the temperature range of 20-93 8C and monitored by changes of the far-UV ellipticity signal at 224 nm. Ramp time was set at 15 min for each temperature increment.…”

Section: Methodsmentioning

confidence: 99%

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Chi

Chien

Liu

et al. 2010

Chemistry A European J

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The antibiotic neocarzinostatin comprises a carrier protein with a well-defined cavity for accommodating an active enediyne chromophore. The protein has two disulfides, one (Cys(37)-Cys(47)) lies on the cavity bottom and the other (Cys(88)-Cys(93)) in a constrained short loop. When the chromophore is not bound to the protein, a thiol-induced cycloaromatization of the enediyne into a tetrahydroindacene derivative is responsible for the potent antitumor activity. When it is protein-bound, the protein diverts the cycloaromatization pathway to form a distinct hydroxyisochromene-type product. How the protein directs the enediyne chemistry is an interesting puzzle, and various suggestions have been proposed in the past. We screened more than fifty thiols and manipulated conditions to locate reaction features and search for factors that could influence the protein directing strength. Thiol- and oxygen-concentration-dependence studies suggested that disulfides, which maintain the steric rigidity of the protein, could play a key role in diverting the cycloaromatization pathway. For direct proofs, we made mutations at each of the two disulfides by replacing sulfur atoms with oxygen. Circular dichroism and two-dimensional NMR spectroscopy studies suggested that the mutations changed neither the protein conformation nor the ligand interactions. Analyses of the thiol-induced cycloaromatization revealed that rupture of Cys(37)-Cys(47) made the protein almost completely lose its chemical directing ability, whereas rupture of Cys(88)-Cys(93) had only a minor influence. The results demonstrated that the steric rigidity of the binding cavity, but not necessary the whole protein, played an important role in the protein-directed mechanism.

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Section: Methodsmentioning

confidence: 99%

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Chi

Chien

Liu

et al. 2010

Chemistry A European J

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Disulfide Bond Substitution by Directed Evolution in an Engineered Binding Protein

et al. 2009

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Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications.The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques.

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Thiols Screened by the Neocarzinostatin Protein for Preserving or Detoxifying its Bound Enediyne Antibiotic

Chi

Huang

Chin

2012

Chemistry A European J

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Neocarzinostatin is an antibiotic chromoprotein produced by Streptomyces carzinostaticus. Its enediyne‐containing chromophore exhibits high DNA cleavage activity and belongs to one of the most potent categories of antitumor agents. The labile chromophore is readily inactivated by environmental thiols including the most abundant glutathione. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are of interest. Site‐directed mutagenesis studies of the neocarzinostatin protein have shown that residues D33 and D99 play primary and secondary roles, respectively, in preserving neocarzinostatin from acidic glutathione whereas D79 and other residues around the opening of the binding cleft have an insignificant effect. Biothiol analyses revealed that cells of S. carzinostaticus produced no glutathione, but instead neutral mycothiol, which is known to serve functions analogous to glutathione. Mycothiol was the only neutral‐charged thiol produced by the organism; all other identified biothiols carried at least partial negative charges. When the bacteria were cultured under conditions that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly, which suggests mycothiol‐dependent cellular detoxification. Treating neocarzinostatin samples with the cell extract that retained active sulfhydryls led to efficient drug inactivation, which indicates that mycothiol is allowed to approach the protein‐bound chromophore. The anionic side‐chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol‐screening operation: Preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge–charge repulsion in the outer‐cell environment and detoxifying the toxin in the inner‐cell body for self‐resistance by accepting the cell‐produced neutral mycothiol.

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Release of the Neocarzinostatin Chromophore from the Holoprotein Does Not Require Major Conformational Change of the Tertiary and Secondary Structures Induced by Trifluoroethanol

Cited by 18 publications

References 44 publications

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Role of Steric Effects in Protein‐Directed Enediyne Cycloaromatization of Neocarzinostatin

Disulfide Bond Substitution by Directed Evolution in an Engineered Binding Protein

Thiols Screened by the Neocarzinostatin Protein for Preserving or Detoxifying its Bound Enediyne Antibiotic

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