N-terminal and C-terminal domains of arrestin both contribute in binding to rhodopsin.
Visual arrestin terminates the signal amplification cascade in photoreceptor cells by blocking the interaction of light activated phosphorylated rhodopsin with the G-protein transducin. Although crystal structures of arrestin and rhodopsin are available, it is still unknown how the complex of the two proteins is formed. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores (Alexa 633). The binding was recorded by time-resolved light scattering. To accomplish site-specific shielding of protein regions, in a first step all three wild-type cysteines were replaced by alanines. Nevertheless, regarding the magnitude and specificity of rhodopsin binding, the protein is still fully active. In a second step, new cysteines were introduced at selected sites to allow covalent binding of fluorophores. Upon attachment of Alexa 633 to the recombinant cysteines we observed that these bulky labels residing in the concave area of either the N- or the C-terminal domain do not perturb the activity of arrestin. By simultaneously modifying both domains with one Alexa 633 the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding.
Skegro
Darko
D
Forschungszentrum Jülich, INB 2/Molekulare Biophysik, Jülich, Germany.
Pulvermüller
Alexander
A
Krafft
Bianca
B
Granzin
Joachim
J
Hofmann
Klaus Peter
KP
Büldt
Georg
G
Schlesinger
Ramona
R
D000818:Animals; D019393:Arrestin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000235:genetics; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism*; D015398:Signal Transduction
D019393:Arrestin; D017931:DNA Primers; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012243:Rhodopsin; D003545:Cysteine