Different Properties of the Native and Reconstituted Heterotrimeric G Protein Transducin

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• 作者：Anna Goc ; Thomas E. Angel ; Beata Jastrzebska ; Benlian Wang ; Patrick L. Wintrode ; Krzysztof Palczewski
• 刊名：Biochemistry
• 出版年：2008
• 出版时间：November 25, 2008
• 年：2008
• 卷：47
• 期：47
• 页码：12409-12419
• 全文大小：492K
• 年卷期：v.47,no.47(November 25, 2008)
• ISSN：1520-4995

文摘

Visual signal transduction serves as one of the best understood G protein-coupled receptor signaling systems. Signaling is initiated when a photon strikes rhodopsin (Rho) causing a conformational change leading to productive interaction of this G protein-coupled receptor with the heterotrimeric G protein, transducin (Gt). Here we describe a new method for Gt purification from native bovine rod photoreceptor membranes without subunit dissociation caused by exposure to photoactivated rhodopsin (Rho*). Native electrophoresis followed by immunoblotting revealed that Gt purified by this method formed more stable heterotrimers and interacted more efficiently with membranes containing Rho* or its target, phosphodiesterase 6, than did Gt purified by a traditional method involving subunit dissociation and reconstitution in solution without membranes. Because these differences could result from selective extraction, we characterized the type and amount of posttranslational modifications on both purified native and reconstituted Gt preparations. Similar N-terminal acylation of the Gtα subunit was observed for both proteins as was farnesylation and methylation of the terminal Gtγ subunit Cys residue. However, hydrogen/deuterium exchange experiments revealed less incorporation of deuterium into the Gtα and Gtβ subunits of native Gt as compared to reconstituted Gt. These findings may indicate differences in conformation and heterotrimer complex formation between the two preparations or altered stability of the reconstituted Gt that assembles differently than the native protein. Therefore, Gt extracted and purified without subunit dissociation appears to be more appropriate for future studies.