There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric
point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein
are involved in electrostatic interactions with negatively charged substrates. We
tested this hypothesis by individually mutating all basic residues in Tlc to Cys.
Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than
80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited
higher than wild-type (WT) activity. At least in one case this up-effect was additive
and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT
protein. Since in these two single mutants and in the corresponding double mutant
K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a
mechanism that limits the transporter's exchange rate and that at least these two
basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc
homologs, the loss of activity in the mutants poorly correlates with charge conservation
within the Tlc family. Also, despite the presence of three positively charged and
one negatively charged intramembrane residues, we have failed to identify potential
charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.