Difference between revisions of "Part:BBa K5079030"

Latest revision as of 02:52, 2 October 2024

Protein Switch 5 (K_45_WT)

In an SDS-PAGE analysis of an enzymatic assay, MS5 demonstrated the production of α-synuclein fragments overtime consistently across both Trial 1 and Trial 2. The observed fragment had an approximate molecular weight of 10 kDa, suggesting cleavage likely occurred near the C-terminus of α-synuclein, possibly at residues D115 or N122. This hypothesis is supported by the fact that KLK6 was inserted into cyclophilin D (Cyp-D) at position K45 in the MS5 variant. K45 is a highly conserved residue among Cyp-D homologs, and its modification may have disrupted the native binding affinity between Cyp-D and α-synuclein. If Cyp-D’s binding to α-synuclein is impaired while KLK6's proteolytic activity remains intact, cleavage at the C-terminus of α-synuclein would be anticipated, which aligns with the experimental observations.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 785
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K5079030 short</partinfo>
-This part's insertion site has a free energy of 1.1. The insertion's residue has a SASA of 57%. The residue is conserved. This protein switch has not gone through circular permutation. This switch does require a linker. The Difference of Ä Between KLK6 N-C Termini Distance and Insertion Loop Diameter is 16 and the loop distance is 5.2. The linker itself is 3 residues long, composed of "GPG".
+In an SDS-PAGE analysis of an enzymatic assay, MS5 demonstrated the production of α-synuclein fragments overtime consistently across both Trial 1 and Trial 2. The observed fragment had an approximate molecular weight of 10 kDa, suggesting cleavage likely occurred near the C-terminus of α-synuclein, possibly at residues D115 or N122. This hypothesis is supported by the fact that KLK6 was inserted into cyclophilin D (Cyp-D) at position K45 in the MS5 variant. K45 is a highly conserved residue among Cyp-D homologs, and its modification may have disrupted the native binding affinity between Cyp-D and α-synuclein. If Cyp-D’s binding to α-synuclein is impaired while KLK6's proteolytic activity remains intact, cleavage at the C-terminus of α-synuclein would be anticipated, which aligns with the experimental observations.
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