ATP-Driven C-Terminal Polypeptide Modification via TeCH-Tag
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Solution Overview
Problem
Existing methods for producing C-terminal thioesters in proteins are limited by the non-multi-turnover nature of intein-mediated splicing and the hydrolysis competition in enzyme-catalyzed thioester synthesis, lacking a robust in vitro method for precise protein structure manipulation.
Innovation Solution
Development of an ATP-driven platform using MccB, a bacterial ubiquitin-activating enzyme, to catalyze C-terminal activation and peptide ligation, enabling high-yield, ATP-dependent protein bioconjugation through O-A M Pylation of polypeptides with diverse C-terminal functional groups, including thioesters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If intein-mediated protein splicing is used to generate C-terminal thioesters, then precise manipulation of protein structure is achieved, but the process cannot be multi-turnover and is not suitable for high-yield production
Solution Approach 1:
The patent introduces an engineered E1-like enzyme as an intermediary catalyst that mediates the formation of C-terminal thioesters through ATP-dependent adenylation. This enzyme acts as a reusable catalyst that can process multiple substrate molecules, replacing the non-multi-turnover intein splicing mechanism while maintaining precision through engineered substrate specificity.
Solution Approach 2:
The patent changes the fundamental reaction mechanism from intein-mediated splicing to E1-like enzyme-catalyzed adenylation followed by thioester formation. This parameter change enables multi-turnover catalysis while maintaining precision through engineered enzyme-substrate recognition, thereby resolving the contradiction between precision and productivity.
2Productivity
If enzyme-catalyzed thioester synthesis is used, then catalytic efficiency is improved, but thioester hydrolysis competes with ligation reducing overall efficiency
Solution Approach 1:
The patent addresses the hydrolysis competition by designing the E1-like enzyme system to rapidly generate thioesters that can immediately undergo ligation. The engineered enzyme provides kinetic control that favors productive ligation pathways while minimizing hydrolysis, converting the potential harm of hydrolysis competition into benefit through rapid catalytic turnover and engineered substrate directing the reaction toward desired products.
3Use of energy by moving object
If ATP-dependent adenylation is used to activate C-terminus, then thermodynamic driving force is provided for peptide bond synthesis, but the method has not been previously applied to in vitro protein bioconjugation
Solution Approach 1:
The patent applies preliminary action by using ATP-dependent adenylation to activate the C-terminus of the substrate protein before the actual ligation reaction occurs. This pre-activation step creates a high-energy intermediate that is primed for efficient peptide bond formation, enabling the thermodynamically favorable ligation reaction to proceed with high efficiency in vitro.
Solution Approach 2:
The patent introduces the E1-like enzyme as an intermediary that facilitates the application of ATP-dependent adenylation to in vitro protein bioconjugation. This enzyme mediator bridges the gap between the thermodynamic advantage of ATP-driven activation and the practical needs of in vitro protein modification, making the method accessible and controllable for biochemical applications.
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
This method mimics biological peptide bond synthesis for precise in vitro manipulation of protein structure, allowing high-yield formation of C-terminal thioesters and diverse functional groups, enhancing protein bioconjugation efficiency.
Implementation Method 1
reacting the polypeptide, the E1-like superfamily enzyme, and ATP under conditions to O-AMPy late the C-terminus of the polypeptide
Implementation Method 2
MccB, a bacterial ubiquitin-activating enzyme, to catalyze C-terminal activation
Implementation Method 3
reacting the C-terminally O-AMPyated polypeptide with a nucleophile comprising a functional group to provide a modified polypeptide comprising the C-terminal functional group
Implementation Method 4
Cleavage of ATP to AMP and pyrophosphate (PPi) provides a large thermodynamic driving force (ΔG°′=−45.6 kj/mol for hydrolysis) for otherwise unfavorable biosynthetic reactions
Data Source
AI summary
A polypeptide fusion comprising a polypeptide having a C-terminus and a Thioesterification C-terminal Handle (TeCH-tag) fused to the C-terminus of the polypeptide, and a method of modifying the C-terminus of a polypeptide using the polypeptide fusion. The TeCH-tag comprises a sequence of formula (X),X′, wherein X is any amino acid, n is an integer from 6 to 55, and X′ is an amino acid other than asparagine. The TeCH-tag is a substrate of an E1-like superfamily enzyme, and the method comprises reacting the polypeptide fusion, the E1-like superfamily enzyme, and ATP under conditions to O-AM Pylate the C-terminus of the polypeptide fusion; and reacting the C-terminally O-AM Pylated polypeptide fusion with a nucleophile comprising a functional group to provide a modified polypeptide fusion comprising the C-terminal functional group.