Constitutive activation of opsin: influence of charge at position 134 and size at position 296.
In previous studies, mutation of Lys296 or Glu113 in opsin has been shown to result in constitutive activation of the protein--that is, these mutants can activate the G protein transducin in the absence of chromophore and in the absence of light. These and other data have led to the suggestion that a salt bridge between Lys296 and Glu113 helps to constrain opsin to an inactive conformation. It is shown here that of 12 different amino acids substituted at position 296, all, except Arg and the wild-type Lys, are constitutively active at neutral pH, lending further support to this suggestion. However, activation of opsin appears also to be influenced significantly by the size of amino acid side chain at position 296. Thus, there are multiple effects of the mutations. Wild-type opsin is also shown to be weakly active at pH 6.1. Five other charged amino acids in the membrane-embedded region of the protein (Asp83, Glu122, Glu134, Arg135, and Glu201) were mutated to see if they affect constitutive activity. Of these amino acids, only mutation of Glu134 results in an increase in the activity of opsin. Changing Glu134 to Gln increases the activity of opsin, while changing Glu134 to Asp inhibits activity. These results suggest that a negative charge on Glu134 is important in stabilizing the inactive state of opsin. Glu134 is highly conserved in all visual pigments and most of the other G protein-linked receptors.
Cohen
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Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Yang
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Robinson
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Oprian
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D000818:Animals; D002460:Cell Line; D004563:Electrochemistry; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D017299:Rod Opsins / Q000737:chemistry* / Q000502:physiology; D013329:Structure-Activity Relationship; D015293:Transducin / Q000378:metabolism
D005971:Glutamates; D017299:Rod Opsins; D018698:Glutamic Acid; D015293:Transducin