Genetically Encoded DAP for Native-Like Acyl Capture
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Solution Overview
Problem
Existing methods struggle to stabilize and characterize acyl-enzyme intermediates formed between sulfhydryl or hydroxyl sidechains of cysteine or serine residues and carbonyl groups in substrates, as these intermediates are unstable with half-lives of minutes to hours, making their characterization challenging.
Innovation Solution
Incorporation of 2,3-diamino propionic acid (DAP) into enzyme active sites using a genetically encoded orthogonal tRNA synthetase, allowing for stable amide bond formation with substrates, enabling the capture of acyl-enzyme complexes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If substrate analogues bearing electrophilic replacements for carbonyl group are used to capture acyl-enzyme intermediates, then the intermediates can be stabilized and characterized, but the active site becomes non-native and requires synthesis of substrate analogues
Solution Approach 1:
The invention changes the chemical parameter of the nucleophilic residue by replacing cysteine or serine with DAP, which has different reactivity characteristics. This allows the formation of stable amide bonds with carbonyl groups while maintaining a native-like active site structure, resolving the contradiction between intermediate stability and ease of obtaining native-like complexes
Solution Approach 2:
DAP acts as an intermediary residue that bridges the gap between the native catalytic mechanism and stable intermediate capture. It provides the necessary nucleophilicity to form stable amide bonds while maintaining structural similarity to the original catalytic residue, enabling both stability and native-like characteristics
2Reliability
If cysteine residues are replaced with lysine to form amide bonds with ubiquitin C-terminus, then insights into ubiquitination pathways can be gained, but the substitution is far from isosteric and requires elevated pH
Solution Approach 1:
The invention changes the chemical parameter of the nucleophilic residue from lysine to DAP, which has pKa and steric properties more similar to cysteine and serine. This allows the reaction to proceed under physiological pH conditions while maintaining the ability to form stable amide bonds, resolving the contradiction between reliability of insights and ease of operation under physiological conditions
Solution Approach 2:
DAP provides local quality improvement by having steric and electronic properties that are more similar to the original catalytic residues (cysteine/serine) compared to lysine. This local optimization allows for more native-like behavior while still enabling stable intermediate capture
3Quantity of substance
If solid phase synthesis/conjugation techniques are used to incorporate DAP into polypeptides, then DAP-containing polypeptides can be produced, but incorporation into enzyme active sites is extremely difficult or impossible
Solution Approach 1:
The invention replaces the mechanical/chemical approach of solid phase synthesis with a biological system (orthogonal translation system). This allows for site-specific incorporation of DAP into enzyme active sites through genetic coding, making the process as easy as standard protein expression and enabling incorporation into locations that were previously inaccessible
Data Source
AI summary
The invention relates to genetic incorporation of 2,3-diamino propionic acid (DAP) into polypeptides, to unnatural amino acids comprising DAP, to a tRNA synthetase for charging tRNA with unnatural amino acids comprising DAP, and to methods of using the resulting polypeptides, for example in capturing substrates and/or intermediates in enzymatic reactions. The invention also relates to compounds of formula (I) or (II):or salts, solvates, tautomers, isomers or mixtures thereof.