Nam Ki Lee

Single-Molecule Approach to Molecular Biology in Living Bacterial Cells

Recent developments on fluorescent proteins and microscopy techniques have allowed the probing of single molecules in a living bacterial cell with high specificity, millisecond time resolution, and nanometer spatial precision. Recording movies and analyzing dynamics of individual macromolecules have brought new insights into the mechanisms of many processes in molecular biology, such as DNA-protein interactions, gene regulation, transcription, translation, and replication, among others. Here we review the key methods of single-molecule detection and highlight numerous examples to illustrate how these experiments are contributing to the quantitative understanding of the fundamental processes in a living cell.

Large α-synuclein oligomers inhibit neuronal SNARE-mediated vesicle docking.

Parkinson disease and dementia with Lewy bodies are featured with the formation of Lewy bodies composed mostly of α-synuclein (α-Syn) in the brain. Although evidence indicates that the large oligomeric or protofibril forms of α-Syn are neurotoxic agents, the detailed mechanisms of the toxic functions of the oligomers remain unclear. Here, we show that large α-Syn oligomers efficiently inhibit neuronal SNARE-mediated vesicle lipid mixing. Large α-Syn oligomers preferentially bind to the N-terminal domain of a vesicular SNARE protein, synaptobrevin-2, which blocks SNARE-mediated lipid mixing by preventing SNARE complex formation. In sharp contrast, the α-Syn monomer has a negligible effect on lipid mixing even with a 30-fold excess compared with the case of large α-Syn oligomers. Thus, the results suggest that large α-Syn oligomers function as inhibitors of dopamine release, which thus provides a clue, at the molecular level, to their neurotoxicity.

Molecular Basis for SMC Rod Formation and Its Dissolution upon DNA Binding

Summary SMC condensin complexes are central modulators of chromosome superstructure in all branches of life. Their SMC subunits form a long intramolecular coiled coil, which connects a constitutive “hinge” dimerization domain with an ATP-regulated “head” dimerization module. Here, we address the structural arrangement of the long coiled coils in SMC complexes. We unequivocally show that prokaryotic Smc-ScpAB, eukaryotic condensin, and possibly also cohesin form rod-like structures, with their coiled coils being closely juxtaposed and accurately anchored to the hinge. Upon ATP-induced binding of DNA to the hinge, however, Smc switches to a more open configuration. Our data suggest that a long-distance structural transition is transmitted from the Smc head domains to regulate Smc-ScpAB’s association with DNA. These findings uncover a conserved architectural theme in SMC complexes, provide a mechanistic basis for Smc’s dynamic engagement with chromosomes, and offer a molecular explanation for defects in Cornelia de Lange syndrome.

Solution single-vesicle assay reveals PIP2-mediated sequential actions of synaptotagmin-1 on SNAREs.

Synaptotagmin‐1 (Syt1) is a major Ca2+ sensor for synchronous neurotransmitter release, which requires vesicle fusion mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). Syt1 utilizes its diverse interactions with target membrane (t‐) SNARE, SNAREpin, and phospholipids, to regulate vesicle fusion. To dissect the functions of Syt1, we apply a single‐molecule technique, alternating‐laser excitation (ALEX), which is capable of sorting out subpopulations of fusion intermediates and measuring their kinetics in solution. The results show that Syt1 undergoes at least three distinct steps prior to lipid mixing. First, without Ca2+, Syt1 mediates vesicle docking by directly binding to t‐SNARE/phosphatidylinositol 4,5‐biphosphate (PIP2) complex and increases the docking rate by 103 times. Second, synaptobrevin‐2 binding to t‐SNARE displaces Syt1 from SNAREpin. Third, with Ca2+, Syt1 rebinds to SNAREpin, which again requires PIP2. Thus without Ca2+, Syt1 may bring vesicles to the plasma membrane in proximity via binding to t‐SNARE/PIP2 to help SNAREpin formation and then, upon Ca2+ influx, it may rebind to SNAREpin, which may trigger synchronous fusion. The results show that ALEX is a powerful method to dissect multiple kinetic steps in the vesicle fusion pathway.

Ab initio studies on the van der Waals complexes of polycyclic aromatic hydrocarbons. II. Naphthalene dimer and naphthalene–anthracene complex

Ab initio calculations were carried out for the naphthalene dimer and naphthalene–anthracene complex to determine their stable geometries and binding energies. Two medium-size basis sets of 6-31G*(0.25) and 6-31+G* were employed at the MP2 level. Five local minima were found for the naphthalene dimer, three of which were parallel-displaced type and the other two T-shaped type. The global minimum geometry was a parallel-displaced structure of a two-layer graphitic type (Ci point group), not the crossed form (D2d). Its energy calculated by the 6-31G*(0.25) and 6-31+G* basis sets was −7.62 and −6.36 kcal/mol, respectively. The naphthalene–anthracene complex showed four local minima, two of which were parallel-displaced type and the other two T-shaped type. The global minimum was a twisted parallel-displaced form (C2), in which the centers of both molecules lie on the same z-axis with their two long axes skewed at an angle of ≈40°. Its energy was −11.30 and −9.52 kcal/mol with the 6-31G*(0.25) and 6-31+G* bas...

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events.

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

Anion clusters of anthracene, Ann−(n=1–16)

We studied the anion clusters of anthracene, Ann− (n=1–16), by mass spectrometry, photoelectron spectroscopy, and theoretical calculations. The magic numbers observed at n=5 and 13 indicated formation of the half-filled and completely-filled first solvation shell, respectively. We found that autodetachment could occur via a short-lived excited state of the anion, producing autodetached electrons at a nearly constant kinetic energy, irrespective of the photon energy. Three distinct forms of anion core previously proposed were confirmed that are monomeric, dimeric, and trimeric in nature. As the clusters grow in size from the monomer, the character of the anion core undergoes multiple switching until the first solvation shell is half-filled. Between the half-filled and completelyfilled first solvation shell, the coexistence of the monomeric and dimeric anion cores was observed at certain cluster sizes, most notably at n=8, 10, and 11. Only the monomeric form of anion core was observed once the first solvati...

Ab initio studies on the van der Waals complexes of polycyclic aromatic hydrocarbons. I. Benzene–naphthalene complex

The stable geometries and binding energies of the benzene–naphthalene complex were studied by the point-by-point method using ab initio calculations at the MP2/6-31G*(0.25) and MP2/6-31+G* levels. Medium-size basis sets were employed not only to save computational time but also to compensate for the tendency of the MP2 method to overestimate the electron correlation energy of aromatic clusters. The use of the 6-31G*(0.25) and 6-31+G* basis sets in the test calculation for the benzene dimer yielded results very similar to those from the CCSD(T) calculation. As for the benzene–naphthalene complex, four stable geometries were found: one parallel-displaced type and three T-shaped ones, with each type similar to the case of the benzene dimer. The global minimum was found to be the parallel-displaced structure whose energy was −4.88 kcal/mol at the MP2/6-31G*(0.25) level and −3.94 kcal/mol at the MP2/6-31+G* level. These values are ∼1.9 times that of the benzene dimer on the same level of calculation. The energ...

Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s–1 between two conformers of a Holliday junction. The rapid diffusional migration of Deinococcus radiodurans single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of Escherichia coli SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution.

Photoelectron spectroscopy of pyrazine anion clusters

We studied the energetics and character of electron binding in the pyrazine anion clusters by mass spectrometry, photoelectron spectroscopy, and theoretical calculations. The mass distribution showed that the minimum number of molecules in a neat cluster of pyrazine to form an anion was two, with a single pyrazine molecule incapable of accommodating an excess electron. On the other hand, even the addition of a very weak solvent such as Ar sufficed to bring the affinity level of pyrazine below the vacuum level. Photoelectron spectra of some pyrazine-containing anion clusters, (Pz)1−–Arn (n=1–10) and (Pz)1−–S1 (S=pyrazine, benzene, and water), were obtained. A vibrational progression was observed in the photoelectron spectra of (Pz)1−–Arn. The electron affinity of pyrazine was determined to be −0.01±0.01 eV from extrapolation. A small drop in incremental electron affinity was observed from (Pz)1−–Ar4 to (Pz)1−–Ar5, indicating closure of the first solvation shell by four Ar atoms. The pyrazine dimer anion wa...