Harmonic analysis of the fluorescence response of bimane adducts of colicin E1 at helices 6, 7, and 10.
J Biol Chem
; 288(7): 5136-48, 2013 Feb 15.
Article
em En
| MEDLINE
| ID: mdl-23264635
ABSTRACT
The pre-channel state of helices 6, 7, and 10 (Val(447)-Gly(475) and Ile(508)-Ile(522)) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Colicinas
/
Escherichia coli
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Canadá