(The) molecular interactions in ribose transporter

Abstract

The high-affinity ribose transporter of Escherichia coli is an ABC transporters, composed of the periplasmic binding protein (RBP or RbsB), the transmembrane component (RbsC) and the ATP-binding protein (RbsA). By the previous genetic study the RbsC has been known to interact with RBP as a homodimer. The molecular interaction of RbsC was further investigated c in this work by in vivo crosslinking and in vitro reconstitution. The RbsC proteins differently labeled with fluorescent dyes were reconstituted into membrane vesicle, and FRET (Fluorescence Resonance Energy Transfer) between two dyes was measured to show that the dyes are located in close proximity. In order to demonstrate that RbsC itself can bind Dribose, intrinsic fluorescence of RbsC in a micelle structure was monitored with an addition Dribose, and Kd of RbsC was determined as 3.8mM. The purified RbsA showed a constitutive ATPase activity, which implies that RbsA need to interact with RbsC for the control of hydrolysis cycle. To study the functional interaction between RBP and RbsC, mutant RBP that is active without RbsA was exploited. The binding of mutant RBP induced an increase in RbsC intrinsic fluorescence while that of wild type RBP decreased the fluorescence. The results implies that the mode of association between mutant RBP and RbsC may be different from that of wild type RBP and RbsC, which is responsible for its RbsA-independent growth phenotype.