期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1824, 期 3, 页码 450-460出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2011.12.009
关键词
Parkinson's disease: leucine-rich repeat kinase 2; Leucine-rich repeat; Protein purification; Homology modeling
资金
- Marie-Curie Reintegration Grant
- FWO Vlaanderen [G.0666.09]
- Katholieke Universiteit Leuven [OT/08/052A]
- Michael J. Fox Foundation
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, (R) under bar as (o) under barf (c) under bar omplex proteins) and kinase domains and a connecting CUR ((C) under bar -terminus (o) under barf (R) under bar OC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain. are not known. In the present study, we modeled the LRRK2 LRR domain (LRRLRRK2) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRRLRRK2 in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRRLRRK2 thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% alpha-helices and 21% beta-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRRLRRK2 multimerization was detected via cross-linking studies. Finally, the LRRLRRK2 clinical mutations did not influence LRRLRRK2 secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRRLRRK2 inter- and intramolecular interactions. (c) 2012 Elsevier B.V. All rights reserved.
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