Engineering heme binding sites in monomeric rop.
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
Giovanna
G
Department of Human and Animal Biology, University of Turin, Turin, Italy.
Di Venere
Almerinda
A
Mei
Giampiero
G
Sadeghi
Sheila J
SJ
Wilson
Jon R
JR
Gilardi
Gianfranco
G
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D004563:Electrochemistry; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; C054243:Rop protein, ColE1 plasmid; D006418:Heme