Source 1primary paper2020Proteins Structure Function and Bioinformatics
Toolkit/Caspase-2 active-site mutants
Caspase-2 active-site mutants
Also known as: created mutants, novel protease, the two mutants
Taxonomy: Mechanism Branch / Component. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Caspase-2 active-site mutants are engineered variants of human Caspase-2 designed in silico to broaden substrate recognition at the substrate N-terminal amino acid position. In vitro experiments confirmed that two proposed mutants showed enhanced promiscuity, including increased recognition of branched amino acids relative to unmutated Caspase-2.
Usefulness & Problems
Why this is useful
These mutants are useful as a rationally engineered protease system for expanding the substrate scope of a caspase-family enzyme. The reported design addresses situations where broader recognition of N-terminal amino acid identities, rather than the native Caspase-2 specificity profile, is desired.
Problem solved
The tool helps solve the problem of narrow active-site specificity in human Caspase-2 by reprogramming the protease to better accommodate alternative substrate side chains. Specifically, the reported mutants increased recognition of branched amino acids and enhanced overall promiscuity toward different N-terminal amino acids.
Taxonomy & Function
Primary hierarchy
Mechanism Branch
Component: A low-level protein part used inside a larger architecture that realizes a mechanism.
Target processes
recombinationImplementation Constraints
The tool consists of active-site mutations introduced into human Caspase-2. Reported development involved in silico design followed by expression of the two proposed mutants and in vitro experimental testing, but the supplied evidence does not specify expression host, purification workflow, assay format, or construct architecture.
The evidence is limited to a single 2020 study and two proposed mutants validated in vitro. The available evidence does not report cellular validation, organism-level performance, quantitative kinetic parameters, or broader benchmarking across diverse substrate panels beyond increased recognition of branched and different N-terminal amino acids.
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Approval Evidence
1 source4 linked approval claimsfirst-pass slug caspase-2-active-site-mutants
we present a novel protease that was designed in-silico to yield enhanced promiscuity toward different N-terminal amino acids... The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results
Source:
computational designsupports
The engineered protease mutants were designed in silico using sequence and structural comparisons of Caspase-2 and Caspase-3 together with free-energy calculations.
These mutations were determined by sequential and structural comparisons of Caspase-2 and Caspase-3 and their effect was additionally predicted using free-energy calculations.
Source:
engineered property improvementsupports
Two active-site mutations in human Caspase-2 increased recognition of branched amino acids relative to the unmutated protease.
Two mutations in the active-site amino acids of human Caspase-2 were determined to increase the recognition of branched amino-acids, which show only poor binding capabilities in the unmutated protease.
Source:
experimental confirmationsupports
In vitro experiments confirmed the simulation results for the two proposed Caspase-2 mutants.
The two mutants proposed in the in-silico studies were expressed and in-vitro experiments confirmed the simulation results.
Source:
substrate scope expansionsupports
Both engineered Caspase-2 mutants showed enhanced activity toward branched amino acids and also toward smaller unbranched amino acids.
Both mutants showed not only enhanced activities toward branched amino acids, but also smaller, unbranched amino acids.
Source:
Comparisons
Source-backed strengths
The engineering strategy combined sequence comparison, structural comparison, and free-energy calculations, providing a mechanistically motivated design route. Two active-site mutants were experimentally expressed and validated in vitro, and both matched the simulation-based prediction of increased substrate promiscuity relative to the unmutated protease.
Ranked Citations
- 1.