Detection of Phosphate Ion and Protein Phosphorylation — Crystal Surfaces, Ionophore Monolayers, and Protein Interactions☆
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Introduction
Several recent works from our research group on molecular recognition and detection of phosphate ions and of protein phosphorylation are reviewed herein. The site/domain or step for the molecular recognition and detection used respectively were crystal surfaces of phosphate salts,1 phosphate-ionophore monolayers for phosphate detection,2 and protein/protein interactions for protein phosphorylation.3
Molecular recognition and detection of inorganic anions and organic anions have been a topic of fundamental and practical importance.4 Molecular recognition of phosphate ions in aqueous solution has hardly been successful, because a highest hydrophilic nature of the phosphate ion makes it difficult for its direct binding with any synthetic receptors in aqueous solutions. In the human body, phosphate plays important roles not only in control of the pH in blood or lymph fluid, but also in the energy and nitrogen metabolism in cells. Moreover, the phosphate analysis in soil and wastewater are indexes of nutrition, environmental monitoring, and biomedical research. The analysis of orthophosphate ion is thus very important in clinical chemistry, environmental chemistry, biochemistry, pharmacology and others. Although many analytical methods for phosphate have been reported, most of inorganic and organic phosphorus compounds are quantified by spectrophotometric methods, using the formation of molybdenum blue. The reason for the fact that this technique is widely adopted is its excellent sensitivity to phosphate. However, on the other hand, the system is complicated and remains unsuitable for application to automated sensing and feedback control. Chemical sensing systems that can continuously and selectively monitor orthophosphate levels in aqueous solutions would therefore be extremely useful; thus, there is a clear need for well-functioning orthophosphate sensors in situ.
Protein phosphorylation by intracellular kinases plays one of the most pivotal roles in signaling pathways within cells (Fig. 1).5 The kinase proteins catalyze transfer of the γ-phosphate of ATP and phosphorylation of hydroxy groups of serines, threonines and/or tyrosines on the substrate proteins. Upon this phosphorylation, the substrate proteins are subject to conformational changes due to negative charges of the phosphates, which subsequently triggers their enzymatic activation and interaction with their respective target proteins. To reveal the biological issues related to the kinase proteins, electrophoresis, immunocytochemistry and in vitro kinase assay have been used. Recently, Bastiaens’ groups have improved an immunofluorescence staining method to detect protein phosphorylation in the cells and tissues.6, 7, 8 However, these conventional methods do not provide enough information about spatial and temporal dynamics of the signal transduction based on protein phosphorylation and dephosphorylation in living cells. In contrast to the kinase signaling, second messenger signaling, such as Ca2+,9, 10, 11 inositol 1,4,5-triphosphate,12 diacylglycerol,13 cyclic AMP14, 15 and cyclic GMP,16, 17 have been visualized using fluorescent indicators in single living cells. The measurements based on those fluorescent indicators have been found to provide a high spatial and temporal resolution enough for dissecting the single cell events of the second messengers.18
Section snippets
Orthophosphate-ion recognition at aqueous surfaces of insoluble orthophosphate salts1
Selective adsorption/desorption processes of the component ions of insoluble inorganic salts at their solid/aqueous interfaces are important in many areas, including analytical chemistry. Among them, the selective adsorption/desorption of component ions of insoluble inorganic salts has been known to cause charge separation at their solid/aqueous interfaces, which is one of the fundamental characteristics of the response mechanism of solid membrane ion-selective electrodes (ISEs).19, 20, 21, 22,
Phosphate ions recognition at monolayers of hydrogen bond-forming receptors2
Hydrogen bonding plays a very important role in molecular recognition of many biological and artificial systems. For example, X-ray crystal structures show that anion binding in various biological systems results from multiple hydrogen bond formation and a size-selective fit of the anion to its host. Also, numerous artificial systems in which hydrogen bonding allows molecular recognition in bulk solutions were studied. However, only in the late 1990s was complementary hydrogen bonding at phase
Fluorescent indicators for imaging protein phosphorylation in single living cells3
To overcome the limitation for investigating the kinase signaling, we developed genetically encoded fluorescent indicators for visualizing the protein phosphorylation in living cells. The principle of the present method is schematically shown in Figure 11. A substrate domain for a kinase protein of interest is fused with a phosphorylation recognition domain via a flexible linker sequence. The tandem fusion unit consisting of the substrate domain, linker sequence and phosphorylation recognition
Conclusions
The crystal surface of orthophosphate salts was revealed to behave as a molecular recognition site for orthophosphate ions. The ng-level amount of selective surface adsorption of analyte ions is sensitively and selectively detected at the substrate QCM without adding any specific precipitating agent in sample solutions. A similar approach was extended to the analysis of sulfate, selenite and l-leucine based on crystal surfaces of their respective salts.56, 57, 58
Highly hydrophilic phosphate ion
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To commemorate Professor Jerry Atwood's 60th birthday.