Calixarene Additives for Membrane Protein Crystallization

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Solution Overview

Problem

Current methods for crystallizing membrane proteins are hindered by the difficulty in obtaining stable, structurally homogeneous populations and the formation of protein-protein contacts, due to the destabilizing effects of detergents and differences in physico-chemical characteristics, leading to limited success in achieving crystalline structures.

Innovation Solution

The use of calix[n]arene derivatives with acid functions and aliphatic chains as crystallization additives, which form supramolecular clusters that intercalate between membrane proteins through ionic interactions, facilitating their crystallization by promoting organized aggregation and cohesion.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Duration of action of stationary object

If detergents are used to extract and stabilize membrane proteins in solution, then the proteins can be kept in solution for several weeks, but the detergents destabilize the structure of membrane domains and prevent protein-protein contacts necessary for crystallization

Engineering Contradiction:
Improvestability of membrane protein in solutionVSAvoidstructural homogeneity of membrane domains
Core Design Contradiction:
Duration of action of stationary objectVSStability of the object's composition

Solution Approach 1:

The patent uses calix[n]arene derivatives as intermediary molecules that mediate between the detergent environment and membrane proteins. These additives have amphiphilic structures with hydrophobic regions that interact with membrane domains and hydrophilic regions that interact with the aqueous environment, thereby stabilizing membrane protein structures without preventing protein-protein contacts for crystallization

Inventive Principle:
Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent modifies the physico-chemical parameters of the crystallization environment by introducing calix[n]arene derivatives with specific properties (molecular size, hydrophobicity, charge). These parameter changes create optimal conditions for membrane protein crystallization by adjusting the balance between solubility and crystallization tendency

Inventive Principle:
Principle #35Parameter changes

2Reliability

If detergents are used to keep membrane proteins in solution, then the proteins remain soluble, but the detergent crowns mask the lipophilic regions and prevent formation of protein-protein contacts

Engineering Contradiction:
Improvesolubility of membrane proteinVSAvoidcrystallization of membrane protein
Core Design Contradiction:
ReliabilityVSEase of manufacture

Solution Approach 1:

The calix[n]arene derivatives act as intermediary molecules that facilitate protein-protein interactions. Their molecular structure allows them to interact with both the detergent crowns and the membrane protein surfaces, bridging the gap and enabling contact between hydrophilic regions of adjacent proteins while maintaining solubility

Inventive Principle:
Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent extracts the problematic detergent molecules from the immediate protein-protein interface by introducing calix[n]arene derivatives that preferentially interact with protein surfaces. This selective extraction of detergents from critical regions allows protein contacts to form while maintaining overall solubility

Inventive Principle:
Principle #2Taking out (Extraction)

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 significantly enhances the crystallization of polar and positively charged molecules, particularly membrane proteins, by creating an environment conducive to crystal formation, as demonstrated by improved crystallization rates and structural determination at the atomic scale.

Implementation Method 1

form supramolecular clusters that intercalate between membrane proteins through ionic interactions

Methodology Applied
Scientific EffectIonic interactions: Ion Repulsion/Attraction

Implementation Method 2

form supramolecular clusters that intercalate between membrane proteins

Methodology Applied
Scientific EffectSupramolecular assembly: Self-Assembly

Implementation Method 3

facilitating their crystallization by promoting organized aggregation and cohesion

Methodology Applied
Scientific EffectCrystallization: Crystallisation

Data Source

PatentEP2416859B9Use of additive for the crystallization of proteins and process
Publication Date: 2014.06.04 CENT NAT DE LA RECH SCI (C N R S)
  • EP2416859B9 patent drawingFigure 1A~1B
  • EP2416859B9 patent drawingFigure 1C~1D
  • EP2416859B9 patent drawingFigure 2~3A

AI summary

The present invention relates to the use and to a process involving at least one calix[n]arene derivative substituted by at least one acid functional group on the upper face and at least one aliphatic chain of variable length on the other face, as an additive for crystallization of a polar and/or positively charged molecule. The use and the process of the present invention have the advantage of enabling, facilitating and/or accelerating the crystallization of polar and/or positively charged molecules, especially of membrane proteins that are in solution or soluble, which had previously proved to be very difficult.