Thermostable Protein Surface Engineering
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Solution Overview
Problem
Current protein engineering methods struggle to predictably combine the structural stability of thermophile proteins with the activity characteristics of mesophile proteins, often resulting in unpredictable outcomes and limited success in creating enzymes with desired thermal and functional properties.
Innovation Solution
A recombinant meso-active thermo-stable protein is designed by replacing non-contiguous amino acids on the substrate-binding and catalytically active surface of a thermophile protein with structurally equivalent residues from a mesophile protein, using a rational engineering approach to create a protein that retains the structural stability of the thermophile and the activity characteristics of the mesophile.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If rational residue replacement is used to combine thermophile structural stability with mesophile activity characteristics, then the structural stability is improved, but the activity characteristics are lost
Solution Approach 1:
The protein surface is segmented into functionally distinct regions: the structural core (retained from thermophile) and the active surface (transplanted from mesophile). This segmentation allows independent optimization of stability and activity characteristics through selective residue replacement at structurally equivalent positions on the protein surface.
Solution Approach 2:
Different regions of the protein are assigned different functional qualities: the interior and structural scaffold maintain thermophile characteristics for stability, while the exterior active surface adopts mesophile characteristics for optimal catalytic activity. This local quality differentiation resolves the contradiction between global stability and local adaptability.
2Adaptability or versatility
If random mutagenesis is used to create protein variants, then diversity is increased, but predictability of outcomes decreases
Solution Approach 1:
Instead of random mutagenesis followed by screening, the invention performs preliminary rational design by identifying structurally equivalent positions on the protein surface and pre-selecting residues from homologous mesophile proteins. This preliminary action based on structural homology and sequence alignment dramatically improves predictability of the outcome while maintaining diversity.
Solution Approach 2:
The active surface residues are copied from a known mesophile protein at structurally equivalent positions rather than generated randomly. This copying approach, guided by structural homology, ensures that the transplanted residues will function correctly while maintaining predictability through the use of evolutionarily validated sequences.
3Adaptability or versatility
If chimeric proteins are created by combining domains from different sources, then functional versatility is improved, but structural integrity is compromised
Solution Approach 1:
Instead of combining entire domains as in traditional chimeric proteins, the invention extracts only the active surface residues from the mesophile protein and transplants them onto the thermophile scaffold. This extraction approach minimizes disruption to the structural integrity while achieving functional versatility.
Solution Approach 2:
The invention transitions from domain-level chimerism (one-dimensional) to residue-level transplantation at structurally equivalent positions (three-dimensional spatial mapping). By using structural homology to map positions in three-dimensional space rather than simple sequence alignment, the method maintains structural integrity while achieving functional diversity.
Data Source
Figure 1A~1C
Figure 2A~2B
Figure 3A~3F
AI summary
The present invention deals with examination of the alterability of part, or whole, of the surfaces of beta sheet-based protein structures, focusing especially on enzymes. The alteration is done by supplanting/transplanting a part, or whole, of the surface of one protein onto the surface of a homologous protein of superimposable polypeptide backbone, by exploiting the structural features of beta sheets to alter only the regions of the surface involved in substrate/ligand binding and catalysis. The transplantation involves replacement of a selected set of non-contiguous residues constituting the surface regions desired to be altered in one enzyme/protein, by a set of non-contiguous residues located at analogous positions in the other enzyme/protein, in a manner that is likely to facilitate folding and function of the new protein formed by combining residues from both enzymes/proteins. The present invention also deals with using this surface engineering approach to selectively combine enzyme/protein characteristics from different domains of life that are not ordinarily combined by natural evolution, such as the creation of novel proteins that retains the bulk of the thermostable scaffold of a thermophile enzyme onto which the active surface of a mesophile homolog is transplanted, so as to create a thermo-stable protein with meso-active functional characteristics of pH and temperature of optimal function.