Single-Chain Coiled Coil Proteins for Concentration-Independent Assembly
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Solution Overview
Problem
The formation of trimeric coiled coil structures is concentration-dependent, making them unstable for pharmaceutical use, and is difficult to produce via recombinant methods, with challenges in creating specific heterotrimeric coiled coil structures due to complex atomic interactions.
Innovation Solution
Development of single-chain triple-stranded alpha-helical coiled coil proteins with flexible linker fragments, which are thermodynamically stable and tolerant to amino acid substitutions, allowing for controlled formation of predefined coiled coil structures without concentration dependence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If trimeric coiled coil structures are formed by association of individual peptide molecules, then the coiled coil structures can form, but the formation is extremely dependent on concentration requiring relatively high concentrations which have adverse effects when applied to pharmaceutical compounds
Solution Approach 1:
The patent combines three separate peptide molecules into a single-chain polypeptide that contains all three heptad repeat sequences (HRS1, HRS2, HRS3) covalently linked together. This merging eliminates the need for intermolecular association while maintaining the triple-stranded coiled coil structure, thereby resolving the concentration dependence issue.
Solution Approach 2:
The single-chain polypeptide is segmented into three distinct heptad repeat sequences (HRS1, HRS2, HRS3) separated by linker sequences (L1, L2). This segmentation allows each repeat to form its own alpha-helical strand while maintaining covalent connectivity, enabling the triple-stranded coiled coil structure to form intramolecularly without concentration dependence.
2Adaptability or versatility
If peptidic oligomeric complexes are used to create heterotrimeric coiled coil structures, then diverse coiled coil types can be formed, but the constituting peptides are difficult to produce via recombinant methods due to complex atomic interactions
Solution Approach 1:
The patent merges multiple peptide sequences into a single recombinant polypeptide chain, which can be produced in a single expression step using standard recombinant DNA technology. This eliminates the need to separately synthesize and purify multiple individual peptides and then assemble them, greatly simplifying manufacturing while maintaining the ability to create diverse heterotrimeric structures.
3Manufacturing precision
If non-identical peptides are mixed to obtain heteromeric coiled coils, then specific heterotrimeric structures can be formed, but a distribution of homo- and heteromeric coiled coils will form depending on thermodynamic fitness making specific structure control technically hard
Solution Approach 1:
By covalently linking the three heptad repeat sequences in a specific order within a single polypeptide chain, the patent ensures that only one specific heterotrimeric configuration can form. The intramolecular association is predetermined by the sequence architecture, eliminating the formation of unwanted homo- or alternative heteromeric structures and achieving complete specificity.
Solution Approach 2:
The single-chain design pre-arranges the three heptad repeat sequences in a predetermined order with specific linker sequences between them. This preliminary structural arrangement guides the folding process to form only the desired heterotrimeric coiled coil configuration, avoiding the need to control complex equilibrium distributions that arise when mixing separate peptides.
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
The single-chain coiled coil proteins exhibit high thermal stability and facilitate the formation of specific heteromeric coiled coils, reducing the risk of undesired associations and enhancing fold specificity, making them suitable for therapeutic, diagnostic, and purification applications.
Implementation Method 1
said protein spontaneously folding in aqueous solution by way of the HRS1, HRS2 and HRS3 fragments forming a triple-stranded, antiparallel, alpha-helical coiled coil structure
Implementation Method 2
at least 50%, 70%, 90%, or 100% of the conventional heptad positions 'a' and 'd' are occupied by amino acids selected from the group consisting of valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan, histidine, glutamine, threonine, serine, alanine derivatives thereof
Data Source
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AI summary
The present invention relates to single-chain proteins of the formula HRS1-L1-HRS2-L2-HRS3, wherein HRS1, HRS2 and HRS3 are heptad repeat sequences and L1 and L2 are structurally flexible linker sequences, and wherein HRS1, HRS2 and HRS3 form a thermodynamically stable triple-stranded, antiparallel, alpha-helical coiled coil structure in aqueous solution. The invention also relates to amino acid sequence variants, conditions and methods to obtain such proteins and variants, and usages thereof, especially their usage as scaffolds and as therapeutic products.