MODEL COMPOUNDS GUIDE AFFINITY MEASUREMENT OF BERYLLIUM AND CALCIUM INTERACTIONS WITH PHOSPHOLIPIDS

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2023

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Abstract

Divalent cations bound to anionic lipids are necessary co-factors for many signaling mechanisms taking place at both the inner and outer surfaces of the cytoplasmic membrane. Coordination of divalent ions jointly by phospholipid headgroups and specific protein domains mediates recognition and triggers secondary messenger cascades or membrane fusion events. Phosphoryl oxygens of phospholipids are common contributors to divalent ion coordination. With the aims of elucidating the affinities of the calcium ion Ca(2+) and its toxic competitor beryllium Be(2+) to different types of phosphate groups taking place in many ‘building blocks’ of the cell, improving simulation force fields and better understanding the nature of beryllium toxicity, here we use isothermal titration calorimetry (ITC) to study the thermodynamic parameters and coordination of these ions by phosphates. Particularly, we focus on the differences between phosphates in the phosphodiester configuration that connect the glycerol backbone with a headgroup (as in phosphatidylglycerol, PG) and terminal monoester phosphates such as in phosphatidic acid (PA) and most phosphorylated proteins. The comparison of small model compounds, dimethyl phosphate (DMP, mimicking phosphate in phosphatidyl glycerol) with glycerol-3-phosphate (Gly3P, emulating phosphate in phosphatidic acid) shows that the affinity of Be(2+) for Gly3P is about one order of magnitude higher than for DMP and may exhibit at least two binding configurations. The Be(2+) -DMP thermograms in most cases are well fitted with a one-site model. Upon completing the survey of small (model) compounds, we performed experiments to compare the binding parameters of Be(2+) to POPA and to POPG-containing liposomes with the parameters obtained on respective model compounds. We also present several pilot binding experiments performed with POPS liposomes; however, the fit is poor.

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