PagP, an outer membrane enzyme of Gram-negative bacteria, transfers a palmitate chain from phospholipids to lipid A to provide bacterial resistance against host immune defences. PagP acyl chain selection is determined by the hydrocarbon ruler. We have methylated the free sulfhydryl group at the floor of the hydrocarbon ruler in PagPG88C by using site-directed chemical labelling. The reaction was shown to be quantitative by using electrospray ionization mass spectrometry and resulted in the expected shift in PagP acyl-chain selection by a single methylene-unit. The detergent-refolded proteins were structurally characterized by circular dichroism spectroscopy, revealing an exciton couplet that was extinguished in the PagPG88C mutant and subsequently restored upon chemical methylation. We demonstrate that a local structural perturbation arising from the PagPG88C sulfhydryl group was associated with the loss of the exciton and a widening of the acyl-chain resolution. Consequently, the exciton reports methylene-unit resolution in the PagP hydrocarbon ruler.