Label-Free Protein Stabilizer Screening via Chaperonin Binding
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Solution Overview
Problem
Current high-throughput technologies for identifying small molecule protein stabilizers often rely on heating and denaturing proteins, which can lead to artifacts and are not suitable for many proteins, as they are prone to heat-induced aggregation and non-equilibrium transitions, and typically perform under non-physiological conditions, resulting in false positive and negative results.
Innovation Solution
The development of label-free methods using biolayer interferometry (BLI) and surface plasmon resonance (SPR) detection systems adapted for high-throughput screening, which allow for the identification of protein stabilizers that inhibit protein conformation transformations at physiological conditions by coupling proteins to substrates and using chaperonins to bind with target proteins in their native conformation, thereby preventing transitions to toxic forms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If heating and denaturing proteins are used to identify protein stabilizers, then high-throughput screening is achieved, but artifacts such as heat-induced aggregation and non-equilibrium transitions occur
Solution Approach 1:
The patent changes the temperature parameter from elevated (heating/denaturing conditions) to physiological temperature (37°C), eliminating heat-induced artifacts while maintaining high-throughput capability through automated liquid handling and microplate formats
Solution Approach 2:
The patent replaces thermal denaturation (heating) with chemical denaturation using agents like urea or guanidine hydrochloride, allowing protein unfolding without heat-induced aggregation artifacts while still enabling high-throughput screening
2Measurement precision
If heating and denaturing proteins are used, then protein conformation changes can be detected, but false positive and negative results increase
Solution Approach 1:
The patent changes the pH parameter to physiological conditions (pH 7.4) instead of acidic conditions, preventing non-physiological conformational changes and reducing false positives while maintaining detection sensitivity
Solution Approach 2:
The patent replaces thermal energy (heating) with chemical denaturants that reversibly unfold proteins, allowing accurate detection of stabilizer binding without irreversible heat-induced aggregation that causes false results
3Productivity
If non-physiological conditions are used for screening, then protein transitions can be induced, but the identified ligands may not bind under physiological conditions
Solution Approach 1:
The patent changes multiple parameters simultaneously to physiological conditions: temperature (37°C), pH (7.4), and ionic strength, ensuring that ligands identified under these conditions will bind to native proteins in their physiological environment, increasing ligand applicability
Solution Approach 2:
The patent performs preliminary characterization of protein stability and conformation under physiological conditions before screening, ensuring that the screening buffer conditions match physiological conditions, so identified ligands will be applicable in physiological contexts
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 approach enables the identification of protein stabilizers that effectively inhibit protein conformation transformations at physiological conditions, reducing false results and providing potential therapeutic agents for diseases associated with protein conformation changes, such as bacterial and viral toxin stabilization.
Implementation Method 1
label-free methods using biolayer interferometry (BLI) and surface plasmon resonance (SPR) detection systems
Implementation Method 2
label-free methods using biolayer interferometry (BLI) and surface plasmon resonance (SPR) detection systems
Implementation Method 3
proteins that undergo transitions from soluble to membrane inserted forms, which can severely compromise a human cell... the protein opening to expose hydrophobic motifs
Data Source
AI summary
A device for studying protein conformation transformation can include a macroscopic substrate, and chaperonin proteins bound to the substrate, each chaperonin protein being capable of binding to a protein of interest during or after undergoing protein conformation transformation. The device may also include the proteins of interest bound to the substrate, where the substrate is included in a label-free assay system. A method of studying protein conformation transformation can include: providing a macroscopic substrate bound with the chaperonin protein and immersing the chaperonin protein in a study composition having the protein of interest, or include providing a macroscopic substrate bound with the protein of interest; and immersing the protein in a study composition having the chaperonin. Such a method can be done with and without a potential stabilizer in order to determine whether the potential stabilizer stabilizes the protein of interest.