Interference of pH buffer with Pb2+-peripheral domain interactions: obstacle or opportunity?

Abstract

ABSTRACTPb2+is a xenobiotic metal ion that competes for Ca2+-binding sites in proteins. Using the peripheral Ca2+-sensing domains of Syt1, we show that the chelating pH buffer Bis-Tris enables identification and functional characterization of high-affinity Pb2+sites that are likely to be targeted by bioavailable Pb2+.Significance to MetallomicsSyt1, a key regulator of Ca2+-evoked neurotransmitter release, is a putative molecular target of Pb2+. We demonstrate that the use of a chelating pH buffer Bis-Tris enables identification of Ca2+-binding sites that would be most susceptible to Pb2+attack in the cellular environment. In addition, experiments conducted in Bis-Tris revealed the differences between the membrane-binding responses of two Ca2+-sensing domains of Syt1, C2A and C2B. This work advances the understanding of how Pb2+interacts with multipartite Ca2+-binding sites, and illustrates that conducting the experiments under both chelating and non-chelating conditions could provide valuable insight into the mechanism of metallosensory proteins.