Difference between revisions of "Part:BBa K4165034"

Revision as of 19:22, 13 October 2022

HtrA1 Switch number 14

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), pGS-21a RBS (BBa_K4165016), 6x His-tag (BBa_K4165020), WAP inhibitor (BBa_K4165008), GSGSG linker (BBa_K4165066), TD28rev (BBa_K4165006), GGSGGGGG linker (BBa_K4165019), WWW (BBa_K4165007), GSGSG linker (BBa_K4165066), H1A (BBa_K4165000) and T7 terminator (BBa_K731721).

Usage and Biology

Switch 14 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 peptide binding PDZ, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 upon clamp binding to the target protein requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.

The TD28REV and WWW peptides considered as tau binding peptides are proved experimentally to bind with tau inhibit the aggregations of tau aggregations respectively. The H1A peptide was also proven to bind with the PDZ of HtrA1 experimentally. The last part is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, it will act and inhibit the Protein. The whole construction was similarly proved from literature. The process of assembly of the whole switch was done according to both CAPRI and NCBI protocols.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 379
Illegal AgeI site found at 115
1000
COMPATIBLE WITH RFC[1000]

Modeling

The switch was modeled by (Alphafold - Rosettafold - tRrosetta) and the top model was obtained from tRrosseta with a score of 4 out of 6 according to our quality assessment code.

cbeta_deviations	clashscore	molprobity	ramachandran_favored	ramachandran_outliers	Qmean_4	Qmean_6
1	4.37	1.46	96.35	0	0.714254	-0.75817

                              Figure 1. The 3D structure of switch 14 model visualized by Pymol

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-Switch 14 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 PDZ peptide, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.
+Switch 14 is used to mediate the activity of HTRA1. It is composed of 3 parts connected by different linkers; an HtrA1 peptide binding PDZ, a clamp of two targeting peptides for tau or amyloid beta, and a catalytic domain inhibitor. Activating HTRA1 upon clamp binding to the target protein requires a conformational change in the linker, eliminating the attached inhibitor from the active site. The conformational rearrangement can be mediated through the binding of affinity clamp to tau or beta-amyloid. This binding will result in a tension that detaches the inhibitor from the active site.
+The TD28REV and WWW peptides considered as tau binding peptides are proved experimentally to bind with tau inhibit the aggregations of tau aggregations respectively. The H1A peptide was also proven to bind with the PDZ of HtrA1 experimentally. The last part is the inhibitor, which is mainly a serine protease inhibitor, and since our protease is a serine protease, it will act and inhibit the Protein. The whole construction was similarly proved from literature. The process of assembly of the whole switch was done according to both CAPRI and NCBI protocols.
 <h2>Sequence and Features</h2>