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Completed Project

Characterizing the Stereochemistry of the Headgroup-Acylated Phospholipid Acyl Phosphatidylglycerol in Escherichia coli

Ciara Tomlinson ’18 and Teresa Garrett (Chemistry)

Acyl phosphatidylglycerol (acyl PG) is a glycerophospholipid found in Escherichia coli. Although found in low levels in E. coli, acyl PG could play a role in the function of the bacteria’s cell membrane. In order to learn more about this lipid’s function, we first have to understand its structure. There are currently multiple possible structures for acyl PG, including four with key stereochemical differences at the sn-2 and sn-2’ positions of the glycerol headgroup. The stereochemistry of acyl PG has yet to be characterized as one of these four, an entirely new structure or a mixture of multiple isoforms. In this study, we used two methods to characterize acyl PG: two dimensional NMR spectroscopy and enzymatic characterization of glycerol stereochemistry. Through 2D correlation spectroscopy (2D COSY), we obtained detailed 1H NMR and 2D COSY spectra of bis(monoacylglycerolphosphates) (BMPs), which are similar to possible structures of acyl PG except BMP lacks the sn-2 acyl chain. The crosspeaks of the 2D COSY spectra were then analyzed to determine whether this method could be used to characterize acyl PG’s stereochemistry directly. Upon comparison analysis of 3,3’ BMP and 1,1’ BMP, no clear distinctions were made between the two BMP standards’ spectra. In future experiments, the number of 1H NMR scans could be increased before being transposed onto 2D COSY spectrum to increase spectrum resolution. The reaction of the enzyme α-glycerophosphate dehydrogenase (α-gpdh) and sn-3-glycerolphosphate (gly3p) converts NAD+ to NADH. However, if presented with sn-1-glycerophosphate the reaction does not occur; it is stereospecific. The reaction was found to go to completion at 37°C when using 10mM NAD+ and limiting concentrations of gly3p as low as 15µM. Next steps include performing the enzymatic assay with acyl PG in place of gly3p and if the conversion of NAD+ occurs, it could indicate the presence of sn-3-glycerolphosphate instead of sn-1-glycerolphosphate in acyl PG.