Structure and function in rhodopsin: Kinetic studies of retinal binding topurified opsin mutants in defined phospholipid-detergent mixtures serve asprobes of the retinal binding pocket
Pj. Reeves et al., Structure and function in rhodopsin: Kinetic studies of retinal binding topurified opsin mutants in defined phospholipid-detergent mixtures serve asprobes of the retinal binding pocket, P NAS US, 96(5), 1999, pp. 1927-1931
Citations number
19
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
In the current standard procedure for preparation of mammalian rhodopsin mu
tants, transfected COS-1 cells expressing the mutant opsin genes are treate
d with 5 mu M 11-cis-retinal before detergent solubilization for purificati
on. We found that binding of 11-cis-retinal to opsin mutants with single am
ino acid changes at Trp-265 (W265F,Y,A) and a retinitis pigmentosa mutant (
A164V) was far from complete and required much higher concentrations of 11-
cis-retinal. By isolation of the expressed opsins in a stable form, kinetic
studies of retinal binding to the opsins in vitro have been carried out by
using defined phospholipid-detergent mixtures. The results show wide varia
tion in the rates of 11-cis-retinal binding. Thus, the in vitro reconstitut
ion procedure serves as a probe of the retinal-binding pocket in the opsins
. Further, a method is described for purification and characterization of t
he rhodopsin mutants after retinal binding to the opsins in vitro.