Disruption of the 5 Helix of Transducin Impairs Rhodopsin-Catalyzed Nucleotide Exchange
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- 作者：Ethan P. Marin ; A. Gopala Krishna ; and Thomas P. Sakmar
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：June 4, 2002
- 年：2002
- 卷：41
- 期：22
- 页码：6988 - 6994
- 全文大小：297K
- 年卷期：v.41,no.22(June 4, 2002)
- ISSN：1520-4995
文摘
Photoactivated rhodopsin (R*) catalyzes nucleotide exchange by transducin, the heterotrimericG protein of the rod cell. Recently, we showed that certain alanine replacement mutants of the 
5 helixof the subunit of transducin (Gt) displayed very rapid nucleotide exchange rates even in the absenceof R* [Marin, E. P., Krishna, A. G., and Sakmar, T. P. (2001) J. Biol. Chem. 276, 27400-27405]. Wesuggested that R* catalyzes nucleotide exchange by perturbing residues on the 5 helix. Here, wecharacterize deletion, insertion, and proline replacement mutants of amino acid residues in 5. In general,the proline mutants exhibited rates of uncatalyzed nucleotide exchange that were 4-8-fold greater thanwild type. The proline mutants also generally displayed decreased rates of R*-catalyzed activation. Thedegree of reduction of the activation rate correlated with the position of the residue replaced with proline.Mutants with replacement of residues at the amino terminus of 5 exhibited mild (<2-fold) decreases,whereas mutants with replacement of residues at the carboxyl terminus of 5 were completely resistantto R*-catalyzed activation. In addition, insertion of a single helical turn in the form of four alanine residuesfollowing Ile339 at the carboxyl terminus of 5 prevented R*-catalyzed activation. Together, the resultsprovide evidence that 5 serves an important function in mediating R*-catalyzed nucleotide exchange. Inparticular, the data suggest the importance of the connection between the 5 helix and the adjacent carboxyl-terminal region of Gt.
5 helixof the subunit of transducin (Gt) displayed very rapid nucleotide exchange rates even in the absenceof R* [Marin, E. P., Krishna, A. G., and Sakmar, T. P. (2001) J. Biol. Chem. 276, 27400-27405]. Wesuggested that R* catalyzes nucleotide exchange by perturbing residues on the 5 helix. Here, wecharacterize deletion, insertion, and proline replacement mutants of amino acid residues in 5. In general,the proline mutants exhibited rates of uncatalyzed nucleotide exchange that were 4-8-fold greater thanwild type. The proline mutants also generally displayed decreased rates of R*-catalyzed activation. Thedegree of reduction of the activation rate correlated with the position of the residue replaced with proline.Mutants with replacement of residues at the amino terminus of 5 exhibited mild (<2-fold) decreases,whereas mutants with replacement of residues at the carboxyl terminus of 5 were completely resistantto R*-catalyzed activation. In addition, insertion of a single helical turn in the form of four alanine residuesfollowing Ile339 at the carboxyl terminus of 5 prevented R*-catalyzed activation. Together, the resultsprovide evidence that 5 serves an important function in mediating R*-catalyzed nucleotide exchange. Inparticular, the data suggest the importance of the connection between the 5 helix and the adjacent carboxyl-terminal region of Gt.