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Using structural information and protein design tools we have done homology modelling and energy calculations to predict the interaction between 20 Ras subfamily proteins with 50 putative Ras binding domains. To validate this network we have cloned six not so well characterized Ras binding domains (RBD) and two Ras proteins (RERG, DiRas1). These, together with previously described RBD domains, Ras and Rap proteins have been analyzed in 70 pull-down experiments. Comparing our interaction network with these and previous pull-down experiments (total of 150 cases) shows very high accuracy for distinguishing between binders and non-binders (~0.80). Bioinformatics information was integrated to sort out those in vitro interactions that are more likely to be relevant in vivo. We proposed several new interactions between Ras family members and effector domains that are of relevance in understanding the physiological role of these proteins.
A general guideline to use homology modeling and energy calculations in order to predict protein interactions will be presented. |
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