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Monitoring the Structural Dynamics of LRRK2 using Split‐ Luciferase Protein‐Fragment‐Assisted Complementation
Author(s) -
Nardozzi Jonathan,
Baker Olivia,
Wong Salinda,
LaVoie Matthew J.
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1013.4
Subject(s) - luciferase , protein fragment complementation assay , lrrk2 , complementation , leucine rich repeat , chemistry , fusion protein , microbiology and biotechnology , kinase , biology , gene , mutation , biochemistry , transfection , mutant , recombinant dna
Missense mutation of the PARK8 gene, which encodes the protein Leucine‐Rich Repeat Kinase 2 (LRRK2), is the most common genetic cause of Parkinson's Disease (PD), yet the physiological role of this protein is largely unknown. LRRK2 dimerization has been linked to its activity and localization in the cell, as dimers have higher kinase activity and are preferentially membrane‐associated. In this study we utilized a Split‐Luciferase Protein‐ Fragment‐Assisted Complementation assay (PCA) to assess dimerization of LRRK2. PCA has numerous advantages over other techniques used to study protein‐protein interactions, as the luciferase reporter displays specificity, reversibility, and signal intensity. We have developed a quantitative bioluminescent assay of LRRK2 dimerization as a means to understanding the regulators of LRRK2 structure and function. Numerous split‐luciferase fragment pairs were explored from both the Renilla and Firefly luciferase reporters, and one fragment pair was optimized for investigating LRRK2 dimerization. Interestingly, we observed that fusion and orientation of luciferase fragments to their respective LRRK2 monomers were important factors for reporter efficiency. Efforts to characterize small molecule regulators of LRRK2 dimerization are ongoing. NIH grant #NS072604