Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Abstract

Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the proteinbound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability. KeywordsEnediyne; Neocarzinostatin; Chromophore; Ligand binding; Holoprotein; Protein stability Recently, much attention has been focused on the protein-ligand conjugation. Most conjugate proteins undergo changes in their conformation and subsequently, in stability on ligand removal [1][2][3]. For example, binding of heme to cytochrome changes the protein conformation and consequently, increases the protein stability [4]. It is rather uncommon for conjugate proteins to adopt a like conformation with or without the ligand. This includes azurin (β protein with a copper ion), flavodoxin (α/β protein with a flavin mononucleotide) [5], neocarzinostatin (NCS, 1 β protein with an enediyne chromophore) [6,7] and so on. In spite of structural similarity, the holo form of azurin is more stable than its apo form [8]. The flavodoxin holo form, in contrast, has little changes in either conformation or stability on its cofactor removal *Corresponding author. Fax: +886 4 22862547. E-mail address: chdhchin@dragon.nchu.edu.tw (D.-H. Chin). 1 Abbreviations used: NCS, neocarzinostatin; holoNCS, neocarzinostatin chromoprotein conjugate complex; apoNCS, apoprotein of neocarzinostatin; NCS-C, neocarzinostatin chromophore; NCS-C(b), protein-bound neocarzinostatin chromophore; CD, circular dichroism; HPLC, high-performance liquid chromatography; EtBr, ethidium bromide; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance; T m , temperature at which half of the protein is unfolded; T r , temperature at which half of the NCS-C(b) is released from NCS complex; C m , concentration of denaturant at which half of the protein is unfolded; C r , concentration of denaturant at which half of the NCS-C(b) is released from NCS com...

Insight into the strong inhibitory action of salt on activity of neocarzinostatin

“…The results were consistent with early reports. 21,22 With salt, no significant changes on the protein stability could be observed at a salt concentration up to 1500 mM. As shown in Figure 2a, unfolding profiles and T m values obtained at 0, 600, and 1200 mM NaCl coincided with each other, indicating unaltered conformational stability of apoNCS under high salt conditions.…”

“…By monitoring changes of the far-UV CD signals at 224 nm, 21 conformational The total lesions included direct strand breaks and abasic site lesions, which were converted to strand breaks after putrescine treatment. All values were averages of minimum 3 repeats and a cap on each bar represents standard deviation.…”

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

“…The CD spectrum of the obtained recombinant WT apoNCS was consistent with previous reports. [30,33,[37][38][39][40] The far-UV CD spectra of WT, C93S, and C37S/C47S apoNCSs ( Figure 5 a) showed a similar negative band around 212 nm, which is indicative of a b-sheet secondary structure in these proteins. The strong positive band of the WT apoNCS centered at 224 nm was somewhat atypical and has been suggested to have some contributions from the two disulfide bonds Cys 37 .…”

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

“…The far-UV CD spectrum of native apoNCS shows a typical negative band at 212 nm for the b-sheet-dominant structure and an atypical positive signal that is centered at 224 nm. [48,[51][52][53][54][55][56] The near-UV CD spectrum, attributed to the characteristic motion of the aromatic side-chains of Y32, W39, and W83 in the tertiary environment of apoNCS, exhibits a weak but broad negative band at around 271 nm. [48,51,[54][55][56] The far-and near-UV CD spectra of mutants F76A, L77A, and S98A apoNCSs (data not shown) are in good agreement with our previous results [48] and closely resemble those of native apoNCS.…”