Heme ligands were introduced in the hydrophobic core of an engineered monomeric ColE1 repressor of primer (rop-S55) in two different layers of the heptad repeat. Mutants rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3) were found to bind heme with a K (D) of 1.1 +/- 0.2 and 0.47 +/- 0.07 microM, respectively. The unfolding of heme-bound and heme-free mutants, in the presence of guanidinium hydrochloride, was monitored by both circular dichroism and fluorescence spectroscopy. For the heme-bound rop mutants, the total free energy change was 0.5 kcal/mol higher in the layer 3 mutant compared with that in the layer1 mutant. Heme binding also stabilized these mutants by increasing the [DGobsH2O] by 1.4 and 1.8 kcal/mol in rop-L63M/F121H and rop-L56H/L113H, respectively. The reduction potentials measured by spectroelectrochemical titrations were calculated to be -154 +/- 2 mV for rop-56H/113H and -87.5 +/- 1.2 mV for rop-L63M/F121H. The mutant designed to bind heme in a more buried environment (layer 3) showed tighter heme binding, a higher stability, and a different reduction potential compared with the mutant designed to bind heme in layer 1.

Di Nardo, G., Di Venere, A., Mei, G., Sadeghi, S.J., Wilson, J.R., & Gilardi, G. (2009). Engineering heme binding sites in monomeric rop. JBIC, 497-505 [10.1007/s00775-009-0465-0].

Engineering heme binding sites in monomeric rop.

DI VENERE, ALMERINDA;MEI, GIAMPIERO;
2009

Abstract

Heme ligands were introduced in the hydrophobic core of an engineered monomeric ColE1 repressor of primer (rop-S55) in two different layers of the heptad repeat. Mutants rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3) were found to bind heme with a K (D) of 1.1 +/- 0.2 and 0.47 +/- 0.07 microM, respectively. The unfolding of heme-bound and heme-free mutants, in the presence of guanidinium hydrochloride, was monitored by both circular dichroism and fluorescence spectroscopy. For the heme-bound rop mutants, the total free energy change was 0.5 kcal/mol higher in the layer 3 mutant compared with that in the layer1 mutant. Heme binding also stabilized these mutants by increasing the [DGobsH2O] by 1.4 and 1.8 kcal/mol in rop-L63M/F121H and rop-L56H/L113H, respectively. The reduction potentials measured by spectroelectrochemical titrations were calculated to be -154 +/- 2 mV for rop-56H/113H and -87.5 +/- 1.2 mV for rop-L63M/F121H. The mutant designed to bind heme in a more buried environment (layer 3) showed tighter heme binding, a higher stability, and a different reduction potential compared with the mutant designed to bind heme in layer 1.
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10
English
Di Nardo, G., Di Venere, A., Mei, G., Sadeghi, S.J., Wilson, J.R., & Gilardi, G. (2009). Engineering heme binding sites in monomeric rop. JBIC, 497-505 [10.1007/s00775-009-0465-0].
Di Nardo, G; DI VENERE, A; Mei, G; Sadeghi, S; Wilson, J; Gilardi, G
Articolo su rivista
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/16083
Citazioni
  • ???jsp.display-item.citation.pmc??? 3
  • Scopus 4
  • ???jsp.display-item.citation.isi??? 4
social impact