4. Photoactivated localization microscopy (PALM) for membrane proteins

Parkinsons disease (PD) is the most popular neurodegenerative disease after Alzheimers disease. -Synuclein, the main component of Lewy body, is believed to be the toxic species in PD. However, the detailed molecular pathogenesis of PD is still unclear. Our group investigate the pathogenic behaviors of -Synuclein oligomers on SNARE-mediated vesicle fusion, i.e., the fundamental process of neurotransmission. For thisanalysis, we utilize ALEX (alternating laser excitation) –FRET technique, which enables efficient observation of the vesicle reaction in the presence of -Synuclein oligomers. In our previous study, we suggested that -Synuclein oligomers inhibit vesicle fusion by blocking SNARE complex formation, which is believed to destruct normal dopamine release in neurons. (Choi and Kim et al. PNAS 110, 4087–4092 (2013)

3. Investigating the pathogenesis of Parkinsons disease on neurotransmission


PALM (photoactivated localization microscopy) is one of the most promising super-resolution techniques that use stochastic photo-activation (or photo–switching) of fluorescent proteins. This method is adequate for studying the localization of the specific target proteins in living cells. However, PALM is generally used for understanding only the static view of biomolecules. Compared with PALM, sptPALM (single-particle tracking PALM) has been developed for studying dynamic behaviors of biomolecules in living cells. In our lab, we are actively pursuing to improve PALM technique for studying the dynamic behavior of membrane proteins in living cells.

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SNARE-mediated membrane fusion is one of the essential processes for many biological functions, such as neurotransmission, transport, and secretion of proteins. To study this SNARE-mediated membrane fusion, in vitro vesicle fusion assay has been used at the bulk level. However, this bulk assay cannot discriminate sub-populations generated during fusion reaction, such as docked, fused, and unreacted vesicles. To overcome the limit of bulk assay, we applied for the first time single-molecule spectroscopy, named alternating-laser excitation (ALEX), on vesicle fusion assay. Our single-vesicle assay based on ALEX has the capability of sorting out of the sub-populations in solution and measuring the kinetics of each sub-population. (Kim and Choi et al. EMBO J. 31, 2144-2155, 2012)

2. Single-vesicle assay in solution by alternating-laser excitation

Revealing the oligomeric forms of proteins in various biological conditions is crucial to understanding the functional roles of proteins in biological processes. To study protein oligomerization, size-exclusion chromatography, small-angle X-ray scattering, etc, have been used at the ensemble level. However, these methods are typically indirect or require milligram quantities of protein samples. Our group developed a novel approach for directly probing the oligomeric forms of proteins and their low resolution quaternary structures in solution with a minimal amount of protein by using single molecule ALEX-FRET. One of the strong advantages of single-molecule ALEX-FRET is that it can analyze the subpopulations of heterogeneous species in solution. For example, we demonstrated that drRecR forms a stable dimer and its oligomeric form is modulated by its own concentration or by the interaction with drRecO. (Kim et al, Chem. Commun.48,1138-1140, 2012)


 1. Single-molecule FRET for protein oligomerization

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