Tryptophan²⁰⁷ is involved in the GTP-dependent conformational switch in the α subunit of the G protein transducin: Chymotryptic digestion patterns of the GTPγS and GDP-bound forms

Abstract

The limited proteolytic pattern of transducin,G _t, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the α_t subunit were identified. The α_t subunit in the GTPγS bound form was cleaved into a major 38 kD fragment, whereas α_t-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The βγ_t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The γ_t subunit was not cleaved and only a small portion of β_t was digested into several fragments. In order to determine which proteolytic fragment of α_t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG _t previously³²P-labeled at Cys³⁴⁷ by petrussis toxin-catalyzed ADP-ribosylation. The³²P-label was mainly associated with the α_t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not³²P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of α_t, at Leu¹⁵ and Leu¹⁹. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of α_t, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp²⁰⁷ in a conformation-dependent manner. Trp²⁰⁷ of α_t-GTPγS was resistant to proteolysis but α_t-GDP and the 38 kD fragments of α_t-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp²⁰⁷ changes when GTP or GTPγS is bound, leading to its inaccessibility to chymotrypsin.