Modified Dipeptide Cleavases for Mild, Selective Peptide Sequencing
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Solution Overview
Problem
Existing methods for peptide and protein degradation, such as Edman degradation, are limited by harsh chemical conditions and lack substrate-specific enzymes for efficient amino acid removal, which can be incompatible with sensitive analysis methods like nucleic acid analysis.
Innovation Solution
Development of modified dipeptide cleavases with specific amino acid substitutions in the substrate binding site of dipeptidyl aminopeptidases, allowing for the selective removal of labeled terminal amino acids or dipeptides from polypeptides, while maintaining compatibility with sensitive analysis methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If Edman degradation is used for peptide sequencing, then stepwise degradation of N-terminal amino acid can be achieved, but harsh chemical conditions (strong acids, high temperature) are required which are incompatible with sensitive analysis methods
Solution Approach 1:
The patent replaces the chemical Edman degradation system with an enzymatic system (dipeptide cleavase). The enzyme catalyzes the hydrolysis of peptide bonds to release dipeptides containing N-terminal amino acids, eliminating the need for harsh chemicals like strong acids and anhydrous TFA while maintaining sequencing capability through stepwise amino acid removal.
Solution Approach 2:
The invention changes the reaction conditions from extreme chemical parameters (high temperature, strong acid concentration) to mild enzymatic parameters (physiological temperature, neutral pH buffer). This parameter transformation enables peptide sequencing under conditions compatible with sensitive nucleic acid analysis methods.
2Productivity
If natural aminopeptidases are used for amino acid removal, then processive elimination of amino acids can occur, but substrate specificity is limited and cannot selectively remove labeled terminal amino acids
Solution Approach 1:
The patent introduces specific amino acid substitutions in the substrate binding site of dipeptidyl aminopeptidase to create localized structural features that recognize and bind labeled amino acids. These local modifications (residues 191, 192, 196, 306, 650) confer specific recognition capability for labeled terminal amino acids while maintaining the enzyme's overall catalytic function for efficient amino acid removal.
Solution Approach 2:
The modified dipeptide cleavase automatically recognizes and selectively removes labeled terminal amino acids or dipeptides from polypeptides through its engineered substrate binding site. The enzyme's modified structure enables it to self-distinguish labeled substrates from unlabeled ones, performing selective degradation without requiring additional separation steps.
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 modified dipeptide cleavases enable efficient and selective removal of labeled amino acids, facilitating protein sequencing and analysis without the use of harsh chemicals, and can be used in kits for targeted polypeptide treatment.
Implementation Method 1
Enzymes that are involved in degradation of peptides and proteins, e.g., aminopeptidases, dipeptidyl peptidases, carboxypeptidases, endopeptidases, and others, hydrolyze peptide bonds
Data Source
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AI summary
Provided herein are modified dipeptide cleavases for removing amino acid(s) from peptides, polypeptides, and proteins. Also provided are methods of using the modified dipeptide cleavases for treating polypeptides, and kits comprising the modified dipeptide cleavase. In some embodiments, the methods and the kits also include other components for macromolecule sequencing and/or analysis.