Accelerated CommunicationBiophysical studies support a predicted superhelical structure with armadillo repeats for Ric-8 |
| Maximiliano Figueroa 1, María Victoria Hinrichs 1, Marta Bunster 1, Patricia Babbitt 2, José Martinez-Oyanedel 1, Juan Olate 1 * |
1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile
2Department of Biopharmaceutical Sciences, California Institute for Quantitative Biosciences, University of California at San Francisco, San Francisco, California
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| email: Juan Olate (jolate@udec.cl) |
*Correspondence to Juan Olate, Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Biológicas, Universidad de Concepción, Casilla 160-C, Concepción, Chile
Funded by:
CONICYT Research; Grant Number: 1090150(JO)
CONICYT Travel Fellowship (MF)
| Ric -8 GEF threading armadillo bioinformatics |
Ric-8 is a highly conserved cytosolic protein (MW 63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been described in mammals, Ric-8A and Ric-8B; each possess guanine nucleotide exchange activity (GEF) on heterotrimeric G-proteins, but with different G subunits specificities. To gain insight on the mechanisms involved in Ric-8 cellular functions it is essential to obtain some information about its structure. Therefore, the aim of this work was to create a structural model for Ric-8. In this case, it was not possible to construct a model based on comparison with a template structure because Ric-8 does not present sequence similarity with any other protein. Consequently, different bioinformatics approaches that include protein folding and structure prediction were used. The Ric-8 structural model is composed of 10 armadillo folding motifs, organized in a right-twisted -alpha super helix. In order to validate the structural model, a His-tag fusion construct of Ric-8 was expressed in E. coli, purified by affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD) and thermostability studies. Ric-8 is approximately 80% alpha helix, with a Tm of 43.1°C, consistent with an armadillo-type structure such as -importin, a protein composed of 10 armadillo repeats. The proposed structural model for Ric-8 is intriguing because armadillo proteins are known to interact with multiple partners and participate in diverse cellular functions. These results open the possibility of finding new protein partners for Ric-8 with new cellular functions. |
Received: 23 January 2009; Revised: 10 March 2009; Accepted: 16 March 2009
10.1002/pro.124
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