Calcium Biosensor with Pentagonal Bipyramidal Binding Site
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Solution Overview
Problem
Current calcium sensors face limitations in accurately monitoring calcium signaling in specific subcellular organelles like the endoplasmic reticulum due to interference from endogenous proteins and perturbation of natural calcium signal pathways, and they struggle to achieve the necessary sensitivity for high concentration calcium measurements.
Innovation Solution
Designing an engineered fluorescent host polypeptide with a metal ion binding site comprising negatively charged residues in a pentagonal bipyramidal geometry, integrated into green fluorescent protein (GFP) to create a calcium biosensor that modulates emission and absorbance signals in response to calcium binding, allowing for real-time concentration measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If commercially available dyes are used to monitor calcium concentration, then calcium binding affinity can be achieved, but the dyes cannot be targeted to specific cell compartments in a predictable amount and cause interference from endogenous proteins
Solution Approach 1:
The patent uses a genetically encoded calcium indicator (GECI) as an intermediary that can be specifically targeted to the endoplasmic reticulum through fusion with an ER localization sequence. This GECI consists of a calcium-binding domain (such as calmodulin or troponin C) fused to a fluorescent protein, allowing specific targeting to ER without interference from endogenous proteins while maintaining predictable subcellular localization and calcium measurement capability
2Object-affected harmful factors
If protein-based calcium sensors are expressed in cells, then specific subcompartment targeting is achieved, but sensitivity for high concentration calcium measurements is insufficient
Solution Approach 1:
The patent modifies the calcium-binding domain to alter its calcium binding affinity parameters, specifically engineering it to have higher affinity for calcium ions. This is achieved through rational design of the binding site geometry (pentagonal bipyramidal coordination) and selection of specific amino acid residues that enhance calcium binding strength, thereby increasing sensitivity for measuring high calcium concentrations in the ER while maintaining specific subcompartment targeting
3Measurement precision
If aequorin is used to monitor calcium responses, then calcium binding detection is achieved, but constant addition of coelenterazine is required which is consumed after each reaction
Solution Approach 1:
The patent employs a fluorescent protein-based calcium indicator that is self-sufficient and does not require continuous addition of external substrates. The fluorescent protein component provides stable, long-lasting fluorescence emission upon calcium binding, eliminating the need for repeated coelenterazine addition required by aequorin-based systems, thereby simplifying the measurement protocol while maintaining calcium response monitoring capability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The calcium biosensor enables accurate, real-time monitoring of calcium concentrations in the endoplasmic reticulum with enhanced sensitivity and specificity, overcoming the limitations of existing sensors and providing a tool for understanding calcium signaling and homeostasis in cellular processes.
Implementation Method 1
Because of the electrostatic nature of calcium binding, charged Asp and Glu occur most often in calcium binding sites. The charge number in the coordination sphere also plays a role in calcium binding affinity.
Implementation Method 2
engineered fluorescent host polypeptide having a metal ion binding site... binding of a metal ion analyte to the molecular recognition motif modulates the emission of a fluorescent signal emitted by the fluorescent host polypeptide
Data Source
Figure 1A~1B
Figure 2A~2B
Figure 3A~3D
AI summary
Analyte sensors, methods for producing and using analyte sensors, methods of detecting and/or measuring analyte activity, detecting pH change, and/or, controlling the concentration of an analyte in a system, are disclosed. Embodiments of the analyte sensors according to the disclosure can provide an accurate and convenient method for characterizing analyte activity, detecting pH change, controlling the concentration of an analyte in a system, and the like, in both in vivo and in vitro environments, in particular in living cell imaging.