Sequential proton NMR assignments and secondary structure of aponeocarzinostatin in solution

Remerowski, M. Lyndsay; Glaser, Steffen J.; Sieker, L.C.; Samy, T. S. Anantha; Drobny, Gary P.

doi:10.1021/bi00488a029

Cited by 24 publications

(20 citation statements)

References 51 publications

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“…One residue in NCS, Pro9, is in a cis-conformation as was reported earlier [24]. It is conserved in the sequences of the homologous proteins and has a cis-conformation also in MCR [32].…”

Section: Resultsmentioning

confidence: 81%

Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution

Teplyakov¹,

Obmolova²,

Wilson³

et al. 1993

European Journal of Biochemistry

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The three-dimensional structure of apo-neocarzinostatin, an antitumour antibiotic protein isolated from Streptomyces curzinostuticus, has been determined by X-ray diffraction at 0.15-nm resolution and refined to R = 17.2%. The crystal structure of neocarzinostatin is similar to that of the related proteins actinoxanthin and macromomycin. It is also in good agreement with the solution structure determined by NMR spectroscopy. The protein molecule consists of a seven-stranded antiparallel P-sandwich and a smaller lobe formed by two P-ribbons. A deep cleft between the two lobes is a putative chromophore binding site. Side chains of Trp39, Leu45, Phe52, Phe78 and the disulphide Cys37-Cys47 aligning the binding cleft in neocarzinostatin suggest the importance of hydrophobic interactions in stabilizing the chromophore molecule. Comparison of the atomic models of neocarzinostatin, actinoxanthin and macromomycin reveals functional residues which might determine specificity towards different chromophores.

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“…One residue in NCS, Pro9, is in a cis-conformation as was reported earlier [24]. It is conserved in the sequences of the homologous proteins and has a cis-conformation also in MCR [32].…”

Section: Resultsmentioning

confidence: 81%

Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution

Teplyakov¹,

Obmolova²,

Wilson³

et al. 1993

European Journal of Biochemistry

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“…Although the CD spectrum of holoNCS containing the chromophore is distinctively different from that of apoNCS (Napier et al, 1979(Napier et al, , 1980(Napier et al, , 1981, a number of structural studies by NMR (Adjadj et al, 1990(Adjadj et al, , 1992aGao, 1992;Gao and Burkhart, 1991;Remerowski et al, 1990;Tanaka et al, 1993) and x-ray crystallography (Kim et al, 1993;Sieker et al, 1976;Teplyakov et al, 1993) suggest that the protein conformation of apo-and holoNCS are almost superimposable. The stabilities of apo-and holoNCS are compared here, to assess the contribution of the bound enediyne group to the conformational stability of the chromoprotein.…”

Section: Comparison Of the Thermal-induced Unfolding Of Apo-and Holoncsmentioning

confidence: 99%

Cold Instability of Aponeocarzinostatin and its Stabilization by Labile Chromophore

Jayachithra

Kumar

et al. 2005

Biophysical Journal

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The conformational stability of aponeocarzinostatin, an all-beta-sheet protein with 113 amino-acid residues, is investigated by thermal-induced equilibrium unfolding between pH 2.0 and 10.0 with and without urea. At room temperature, the protein is stable in a pH range of 4.0-10.0, whereas the stability of the protein drastically decreases below pH 4.0. The thermal unfolding of aponeocarzinostatin is reversible and follows a two-state mechanism. By two-dimensional unfolding studies, the enthalpy change, heat capacity change, and free energy change for unfolding of the protein are estimated. Circular dichroism profiles suggest that this protein undergoes both heat- and cold-induced unfolding. The ellipticity changes at far- and near-UV circular dichroism suggest that the tertiary structure is disrupted but the secondary structure remains folded at low temperatures. Interestingly, the labile enediyne chromophore, which is highly stabilized by the protein, is able to protect the protein against cold-induced unfolding, but not the heat-induced unfolding.

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“…The disulfide Cys 88 -Cys 93 is located in a highly constrained, short loop between two b strands, whereas Cys 37 -Cys 47 lies on the bottom of the binding cavity and underneath the triple bond of the enediyne core, within van der Waals contact distance. This particular arrangement suggests the importance of disulfide bonds in interacting with NCS-C. [10,[14][15][16][17][18] Interestingly, the novel chemistry of enediyne cycloaromatization is protein dependent. Without being protein-bound, the reaction is initiated by a thiolate attack at C12 of NCS-C, followed by nucleophilic opening at the epoxide ring (Scheme 1, path A).…”

Section: Introductionmentioning

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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Sequential proton NMR assignments and secondary structure of aponeocarzinostatin in solution

Cited by 24 publications

References 51 publications

Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution

Crystal structure of apo‐neocarzinostatin at 0.15‐nm resolution

Cold Instability of Aponeocarzinostatin and its Stabilization by Labile Chromophore

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

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