EF-hand Polypeptide Chelators for Intracellular Calcium Control
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Solution Overview
Problem
Current methods for manipulating calcium signaling, particularly intracellular calcium concentration, lack cellular specificity and are plagued by side effects, as they either block calcium influx or rely on optogenetics that cannot manipulate endogenous calcium variations or target internal calcium stores effectively, especially in vivo applications where light stimulation is challenging.
Innovation Solution
Development of polypeptides comprising two calcium-binding domains with different affinities linked by a peptide linker, derived from EF-hand motifs, which directly interact with calcium to chelate intracellular calcium, allowing for targeted manipulation of calcium-dependent pathways and cellular processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If pharmacological strategies are used to block calcium influx or chelate extracellular/intracellular calcium, then calcium signaling can be blocked, but cellular specificity is lost and side effects occur
Solution Approach 1:
The invention uses calcium-binding domains with different calcium affinity values to create polypeptides with localized and differential calcium binding properties. This allows specific calcium chelation in particular cellular compartments or signaling pathways while preserving calcium signaling in other regions, thereby achieving cellular specificity and reducing side effects.
Solution Approach 2:
The invention modifies calcium binding parameters by combining domains with different affinity values (Kd ranges from nM to μM). This parameter differentiation enables selective manipulation of calcium signals at different concentration levels and temporal dynamics, allowing precise control over which calcium pathways are affected without broadly disrupting all calcium signaling.
2Reliability
If optogenetics is used to increase intracellular calcium, then electrical activity in excitable cells can be controlled, but endogenous calcium variations cannot be manipulated and light stimulation is technically challenging in vivo
Solution Approach 1:
Instead of using optogenetics to increase intracellular calcium (as in prior art), this invention employs calcium-binding domains that actively chelate and reduce intracellular calcium concentrations. This inverse approach allows manipulation of endogenous calcium variations and internal calcium stores without requiring light stimulation, enabling in vivo applications.
Solution Approach 2:
The invention replaces the optogenetic mechanical/light-based system with a biochemical calcium chelation system. By using polypeptides with engineered calcium-binding domains, the system achieves calcium manipulation through molecular interactions rather than light stimulation, eliminating the technical challenges of in vivo light delivery while enabling control over endogenous calcium dynamics.
3Adaptability or versatility
If calcium-binding domains with different affinities are associated, then the range of calcium-dependent pathways affected is enlarged, but polypeptide structure complexity increases
Solution Approach 1:
The invention segments the calcium-binding function into multiple independent domains (e.g., EF-hand motifs from different calcium-binding proteins) with distinct affinity characteristics. Each domain operates semi-independently, allowing the polypeptide to bind calcium across a broad concentration range through modular domain combinations, thereby enlarging the scope of affected calcium pathways while maintaining manageable structural organization.
Solution Approach 2:
The invention creates composite calcium-binding polypeptides by fusing multiple calcium-binding domains from different sources (e.g., calmodulin, parvalbumin, troponin C) into a single polypeptide chain. This composite structure integrates domains with different calcium affinity values, enabling the polypeptide to function as a multi-affinity calcium sensor or chelator that can respond to diverse calcium signaling events.
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 polypeptides effectively reduce intracellular calcium concentration, preventing downstream effector activation and allowing for precise manipulation of calcium signaling, thereby offering a more specific and effective approach compared to existing methods, with potential applications in treating pathologies associated with intracellular calcium dysregulation.
Implementation Method 1
the first calcium-binding domain and the second calcium binding domain each comprise at least one calcium-binding site derived from a EF-hand motif
Data Source
AI summary
The disclosure pertains to the field of molecular means capable of binding calcium, in particular peptides which are calcium chelators, appropriate for use in vitro or in vivo and preferably capable of targeting specific cellular compartments. Polypeptide comprising a first calcium-binding domain, a peptide linker and a second calcium binding domain, wherein the first and second binding domains are linked through the peptide linker, and wherein: the first calcium-binding domain and the second calcium binding domain each comprise at least one calcium-binding site derived from a EF-hand motif; and, the first calcium-binding domain and the second calcium binding domain differ in at least one calcium-binding site.