Motivation: A basic problem of structural biochemistry studies is to which extent a mutation will affect the stability, and then the function of the protein. From this point of view, an important aspect regards the function of metabolite mitochondrial carriers, in relation to the role that such proteins cover in some mitochondrial pathologies. Sequence studies have shown that the PX(D/E)XX(K/R) signature is characteristic of all mitochondrial carriers, and possibly involved in the transition from the open to closed states, corresponding to the active/inactive state of the carrier. In this study our approach is to combine predictions and experimental validation adopting as a test case the dicarboxylate carrier (DIC) of S. cerevisiae. Because DIC structure is unknown, we integrated a routinely expert dependent strategy in a automatic tool based on a Grid infrastructure to facilitate the generation of carrier models, including site directed mutagenesis. Methods: Transport activity of mutagenized proteins was measured in vitro in reconstituted systems. In parallel in silico experiments protein stability was predicted by using I-Mutant3, available at http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/IMutant3.0. cgi. DIC structure was computed by homology modelling using a service that integrates several software and data involved in a Grid infrastructure, available at https://sara.unile.it/cgi-bin/bioinfo/enter. Results: Protein stability changes upon mutations can be both predicted and/or tested in vitro, by monitoring the function of mutagenized proteins in reconstituted systems. We found that mutations in the DIC carrier signature reduced or blocked nearly completely the protein transport activity, in agreement with the functional role of the carrier motif. Then the question is whether the observed effect on the activity can be also related to a change on protein stability upon mutation. This was tackled by computational methods. According to our predictions protein destabilization would correlate with the observed loss of protein activity. Our data therefore corroborate the finding that single point mutations may hamper protein stability when placed in functionally relevant structural position, and further add to the possibility of predicting a priori whether the mutation destabilize the protein.
Ferramosca A., Mirto M., Tasco G., Tartarini D., Zara V., Aloisio G., et al. (2008). The effect of single mutations on the carrier activity of the dicarboxylate carrier (DIC) of S. cerevisiae: in vitro validation of predictions of protein stability changes. MARTINA FRANCA (TA) : s.n.
The effect of single mutations on the carrier activity of the dicarboxylate carrier (DIC) of S. cerevisiae: in vitro validation of predictions of protein stability changes
TASCO, GIANLUCA;CASADIO, RITA
2008
Abstract
Motivation: A basic problem of structural biochemistry studies is to which extent a mutation will affect the stability, and then the function of the protein. From this point of view, an important aspect regards the function of metabolite mitochondrial carriers, in relation to the role that such proteins cover in some mitochondrial pathologies. Sequence studies have shown that the PX(D/E)XX(K/R) signature is characteristic of all mitochondrial carriers, and possibly involved in the transition from the open to closed states, corresponding to the active/inactive state of the carrier. In this study our approach is to combine predictions and experimental validation adopting as a test case the dicarboxylate carrier (DIC) of S. cerevisiae. Because DIC structure is unknown, we integrated a routinely expert dependent strategy in a automatic tool based on a Grid infrastructure to facilitate the generation of carrier models, including site directed mutagenesis. Methods: Transport activity of mutagenized proteins was measured in vitro in reconstituted systems. In parallel in silico experiments protein stability was predicted by using I-Mutant3, available at http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/IMutant3.0. cgi. DIC structure was computed by homology modelling using a service that integrates several software and data involved in a Grid infrastructure, available at https://sara.unile.it/cgi-bin/bioinfo/enter. Results: Protein stability changes upon mutations can be both predicted and/or tested in vitro, by monitoring the function of mutagenized proteins in reconstituted systems. We found that mutations in the DIC carrier signature reduced or blocked nearly completely the protein transport activity, in agreement with the functional role of the carrier motif. Then the question is whether the observed effect on the activity can be also related to a change on protein stability upon mutation. This was tackled by computational methods. According to our predictions protein destabilization would correlate with the observed loss of protein activity. Our data therefore corroborate the finding that single point mutations may hamper protein stability when placed in functionally relevant structural position, and further add to the possibility of predicting a priori whether the mutation destabilize the protein.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
