Biochemical and biophysical studies on bacterial ABC transporters

Ruchika, Purdue University

Abstract

ABC transporters constitute a large superfamily of multidomain proteins that use ATP as an energy source to power the transport various molecules across the membrane. The E. coli maltose transporter has long served as a prototype to understand the molecular mechanism of this superfamily. MalG511, a binding protein independent mutant of the maltose transporter shows a characteristic basal activity and an interesting biphasic behavior, with low levels of maltose binding protein (MBP) stimulating transport activity while higher levels inhibit transport activity. Remarkably, restoring effect of MBP suppressor mutants obtained at high, inhibitory concentrations of MBP turns it into an attractive model to understand regulatory mechanisms in disease related ABC transporters which show high basal activities, as in P-glycoprotein and CFTR. The events in the catalytic cycle of MalG511, in presence and absence of MBP and suppressor mutant, have been studied by site directed spin labeling EPR spectroscopy. Vanadate trapping experiments revealed a closed transition state, indicating MalG511 uses the same overall mechanism of transport as other ABC transporters. However, we found that the MalG511 mutation shifted the equilibrium towards the semi-open state of the transporter that has higher affinity for MBP; this explains the inhibitory role of MBP at higher concentrations. An essential ABC like transporter, FtsEX, and its interacting partner, an essential amino hydrolase, PcsB, have been characterized from a serious respiratory pathogen, Streptococcus pneumoniae. The ATPase activity of FtsEX has been proposed to regulate hydrolase activity of PcsB through an interaction between extracellular loop of FtsX and coiled coil domain of PcsB. FtsX and FtsE have been individually expressed and purified. Purification of FtsX and its reconstitution into nanodiscs as a dimer has also been optimized successfully. Glutaraldehyde crosslinking also confirmed the imeric state of FtsX. FtsE has been characterized for ATP binding and hydrolysis. Early steps toward producing the complex for biophysical studies have been taken by demonstrating that FtsE can be pulled down by coexpressing with His tagged FtsX.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biochemistry|Biophysics

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