Conformationally Stabilized Ubiquitin Proteins for USP7 Binding
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Solution Overview
Problem
Current methods lack effective means to stabilize conformational states of ubiquitin proteins, which are crucial for specific protein interactions and enzymatic functions, leading to inefficient recognition and modulation by binding partners.
Innovation Solution
Conformationally stabilized ubiquitin proteins with specific amino acid substitutions, particularly in the β1/β2 loop region, are developed to exhibit enhanced binding affinity and slower conformational dynamics, allowing for higher interaction with deubiquitinases like USP7 and inhibiting their activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type ubiquitin protein is used, then natural conformational dynamics are maintained, but binding affinity to deubiquitinases is insufficient
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (e.g., A7C, A8C, A13C, A34C, A36C, A69C, A71C) to alter the conformational parameters of ubiquitin. These substitutions stabilize specific conformational states (particularly the β1/β2 loop region) to enhance binding affinity to deubiquitinases like USP7, while maintaining the protein's overall structure and function.
2Reliability
If conformational stabilization is achieved through amino acid substitutions, then binding affinity increases, but protein flexibility decreases
Solution Approach 1:
The patent applies local quality by introducing amino acid substitutions at specific local positions (particularly in the β1/β2 loop region and other key residues) to stabilize conformational states that enhance binding affinity. These localized changes do not rigidify the entire protein but rather stabilize specific regions while maintaining overall flexibility needed for biological function.
3Reliability
If conformational dynamics are slowed, then specific conformational states are stabilized for better recognition, but natural protein function may be compromised
Solution Approach 1:
The patent applies partial action by introducing a limited number of specific amino acid substitutions rather than extensive modifications. This partial stabilization of conformational states enhances recognition by deubiquitinases while preserving sufficient conformational dynamics and enzymatic activity for the protein to maintain its natural biological functions.
Data Source
AI summary
Provided herein are conformationally stabilized ubiquitin proteins and methods for using the same to identify agents that bind to the stabilized ubiquitin protein or that bind to a protein that interacts with or processes the stabilized form of the ubiquitin protein. Also provided herein are methods for screening for conformationally stabilized proteins having increased binding affinity to a binding partner in comparison to the binding affinity of the wildtype form of the protein to the binding partner.