Cytosolically-Active Peroxidase Mutants for Electron Microscopy
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Solution Overview
Problem
Electron microscopy lacks a robust and sensitive fluorescent protein equivalent for highlighting specific proteins of interest, with existing reporters like Horse Radish Peroxidase (HRP) being limited to specific cellular compartments and prone to inactivation due to strong fixation, and miniSOG being restricted to small fields of view.
Innovation Solution
Development of ascorbate peroxidase (APX) mutants that are cytosolically active and can convert substrates into signal-releasing products, such as osmiophilic polymers or fluorescent dyes, allowing for imaging in various subcellular compartments, including the cytosol and mitochondria, through the expression of Class I heme peroxidase enzymes and their fusion proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If Horse Radish Peroxidase (HRP) is used as an EM reporter, then ease of operation is improved, but applicability is worsened because it only works in the secretory pathway and is inactive in the cytosol
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in HRP (changing cysteine residues to serine or alanine) to eliminate disulfide bonds. This chemical modification changes the enzyme's structural parameters, allowing it to remain active in the reducing environment of the cytosol while maintaining its peroxidase function for EM imaging.
Solution Approach 2:
The patent segments the HRP enzyme structure by removing the disulfide bond connections through mutation. This structural segmentation allows the enzyme to function independently in different cellular compartments without requiring the intact disulfide bond structure that limits wild-type HRP to secretory pathways.
2Measurement precision
If strong fixation is employed in EM, then measurement precision is improved, but reliability is worsened because reporters are prone to inactivation
Solution Approach 1:
The patent applies beforehand cushioning by pre-modifying the HRP enzyme structure through mutation to remove disulfide bonds before fixation. This structural preparation cushions the enzyme against the harsh oxidizing conditions of strong fixation, allowing it to maintain catalytic activity throughout the EM sample preparation process while still achieving high-resolution imaging.
3Measurement precision
If miniSOG is used as an EM reporter, then measurement precision is improved for small fields of view, but productivity is worsened due to limited field of view and complex requirements
Solution Approach 1:
The patent applies universality by creating a mutated HRP enzyme that can function as an EM reporter in multiple cellular compartments (cytosol, mitochondria, secretory pathway) and under various fixation conditions. This multi-functional enzyme replaces the need for compartment-specific reporters and simplifies the imaging workflow, improving productivity while maintaining precision.
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
Enables sensitive and robust microscopy imaging by maintaining enzymatic activity after fixation and allowing for detailed visualization of proteins in multiple cellular compartments, providing high contrast and resolution for electron microscopy.
Implementation Method 1
contacting the sample with a substrate of the Class I heme peroxidase to allow conversion of the substrate into a product via an oxidation reaction catalyzed by the Class I heme peroxidase
Implementation Method 2
both generating contrast by catalyzing the polymerization of a diaminobenzidine (DAB) into an osmiophilic polymer
Implementation Method 3
converted various enzyme substrates (e.g., DAB and Amplex Red) into signal-releasing products (e.g., osmiophilic polymers and fluorescent dyes)
Data Source
AI summary
An imaging method comprising expressing in cells a Class I heme peroxidase, which optionally is fused with a protein of interest or a cellular localization signal peptide, and contacting the cells with a substrate of the Class I heme peroxidase to allow conversion of the substrate into a product via an oxidation reaction catalyzed by the Class I heme peroxidase, wherein the product releases a signal detectable by a microscope such as an electron microscope. Also disclosed herein are monomeric mutants of a Class I heme peroxidase and mutants of the enzyme that exhibit elevated enzymatic activity as compared to the corresponding wild-type counterpart.