Part:BBa_K4016005

zdk

ZDark, a ~ 6 kDa z-affibody developed by the Hahn group, an engineered small protein that binds selectively only to the dark state of LOV2, the photosensory domain from Avena sativa phototropin

Usage and Biology

Light-sensing proteins (LSPs) have been used extensively in optogenetics and other fields to trigger specific subcellular events with light. Upon irradiation with light, the LSP domain undergoes a conformational shift, often revealing a cryptic site or inducing dimerization with a binding partner, thereby triggering downstream effects. LSPs typically absorb light in the visible to infrared wavelengths, and, critically, can undergo their lighttriggered conformational change repeatedly without functional degradation of the protein.

LOVTRAP consists of the blue light-absorbing LOV2 domain of Avena sativa10 and its binding partner ZDark (Zdk). Upon irradiation with light (400– 500 nm), the flavin cofactor becomes excited, allowing it to form a covalent adduct with a cysteine residue in the LOV2 globular domain. This adduct interaction initiates the unraveling of the C-terminal Jα helix, characteristic of LOV2’s excited state. Thermal relaxation allows the cysteine adduct to unbind the flavin cofactor and the Ja helix to re-coil and dock with the main protein body, thereby reverting LOV2 to its dark state. Zdk binds LOV2’s dark state with a dissociation constant (Kd) of 26.2 nM, the highest affinity of any LSP system currently in use, and then unbinds LOV2 upon blue light irradiation (Kd> 4 uM).

LOVTRAP is particularly useful because it can be readily applied to a broad range of proteins and protein activities, and because it enhances the dynamic range, or lit-dark activity difference, for the targeted proteins.

Characterization

This part is validated through four ways: enzyme cutting, PCR, Sequence, and functional testing

PCR

The PCR is performed with Premix EX Taq.

Upper-Prime: 5’-AAGAGAAAGGTGGAGGCCAGTgtggataacaaattcaataaa-3’

Lower-Prime: 5’- TCTCTTCTTCTTGGGACTGGCgctaccaccagaaccaccttt-3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.

Sequence

This part is sequenced as correct after construction.

Experimental Validation

Result

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1] Hui Wang,Marco Vilela,Andreas Winkler,Miroslaw Tarnawski,Ilme Schlichting,Hayretin Yumerefendi,Brian Kuhlman,Rihe Liu,Gaudenz Danuser,Klaus M Hahn. LOVTRAP: an optogenetic system for photoinduced protein dissociation[J]. Nature Methods: Techniques for life scientists and chemists,2016,13(9):

[2] Joshua A. Hammer,Anna Ruta,Jennifer L. West. Using Tools from Optogenetics to Create Light-Responsive Biomaterials: LOVTRAP-PEG Hydrogels for Dynamic Peptide Immobilization[J]. Annals of Biomedical Engineering,2020,48(prepublish):

[3] Hui Wang,Klaus M. Hahn. LOVTRAP: A Versatile Method to Control Protein Function with Light[J]. Current Protocols in Cell Biology,2016,73(1):