Vertebrate opsins belonging to different classes vary in constitutively active properties resulting from salt-bridge mutations.
Vertebrate opsins are classified into one of five classes on the basis of amino acid similarity. These classes are short wavelength sensitive 1 and 2 (SWS1, SWS2), medium/long wavelength sensitive (M/LWS), and rod opsin like 1 and 2 (RH1, RH2). In bovine rod opsin (RH1), two critical amino acids form a salt bridge in the apoprotein that maintains the opsin in an inactive state. These residues are K296, which functions as the chromophore binding site, and E113, which functions as the counterion to the protonated Schiff base. Corresponding residues in each of the other vertebrate opsin classes are believed to play similar roles. Previous reports have demonstrated that mutations in these critical residues result in constitutive activation of transducin by RH1 class opsins in the absence of chromophore. Additionally, recent reports have shown that an E113Q mutation in SWS1 opsin is constitutively active. Here we ask if the other classes of vertebrate opsins maintain activation characteristics similar to that of bovine RH1 opsin. We approach this question by making the corresponding substitutions which disrupt the K296/E113 salt bridge in opsins belonging to the other vertebrate opsin classes. The mutant opsins are tested for their ability to constitutively activate bovine transducin. We demonstrate that mutations disrupting this key salt bridge produce constitutive activation in all classes. However, the mutant opsins differ in their ability to be quenched in the dark state by the addition of chromophore as well as in their level of constitutive activation. The differences in constitutive activation profiles suggest that structural differences exist among the opsin classes that may translate into a difference in activation properties.
Nickle
Benjamin
B
Biological Sciences Department, University of Maryland, Baltimore County, Baltimore, Maryland 21250, USA.
Wilkie
Susan E
SE
Cowing
Jill A
JA
Hunt
David M
DM
Robinson
Phyllis R
PR
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D017299:Rod Opsins / Q000235:genetics / Q000502:physiology*; D015293:Transducin / Q000378:metabolism; D015027:Zebrafish
D017299:Rod Opsins; D015293:Transducin