SangYoon Chung

Structures and Electronic Spectra of CdSe−Cys Complexes: Density Functional Theory Study of a Simple Peptide-Coated Nanocluster

We present density functional theory (DFT) structures and time-dependent DFT electronic excitation energies of several small CdSe nanoclusters with the composition Cd(n)Se(n) (n = 3, 6, 10, 13). We examine the effects on the geometries and excitation spectra of the nanoclusters induced by two chemical changes: peptide-binding and ligand passivation. We use cysteine (Cys) and cysteine-cysteine dipeptide (Cys-Cys) as model peptides and hydrogen atoms as surface-bound solvent ligands (or stabilizing agents). By comparing the results calculated for bare, hydrogen-passivated (Cd(n)Se(n)H(2n)), as well as the corresponding Cys- and Cys-Cys- bound clusters (Cd(n)Se(n)-, Cd(n)Se(n)H(2n), -Cys, -Cys-Cys), we find that peptide-binding blue shifts the electronic excitations of bare nanoclusters, but red shifts those of hydrogen-passivated nanoclusters. The carboxyl oxygen and the sulfur atom tend to form a four-centered ring with adjacent two Cd atoms when the CdSe cluster forms covalent bonds with Cys or Cys-Cys. Further, this type of bonds may be distinguishable by significant red shifts of the excitation energies.

Enzymatically Incorporated Genomic Tags for Optical Mapping of DNA-Binding Proteins

Affordable DNA sequencing is revolutionizing genetic research and is enabling multiple novel biomedical applications. Among the inherent properties of today’s high-throughput sequencing technologies is the fact that it compiles long-range sequences from the assembly of numerous short-read data.[1] This leads to two fundamental limitations: loss of long-range contextual information on the single-genome level and difficulties coping with repetitive or variable genomic regions. Optical mapping and its variants[2–10] rely on the visualization of individual, long (50 kb–1000 kb) DNA molecules and extraction of genomic information by fluorescent labeling of the DNA. These techniques lack the resolution of sequencing but offer genomic context and therefore are attractive both in combination with sequencing to aid in sequence assembly[11–13] and for investigation of genomic structural variations on the individual chromosome level.[14, 15] Such variations include deletions, duplications, copy-number variants (CNVs), insertions, inversions, and translocations, all of which have a major impact on the phenotypic variations within a population (or somatic mutations, important in cancer progression). In addition, the available information content of the genome extends beyond the sequence, and the long-range data offered by optical mapping may provide crucial information regarding the distribution of DNA-binding proteins such as transcription factors and histones along the genome.

Adsorbate-induced absorption redshift in an organic-inorganic cluster conjugate: Electronic effects of surfactants and organic adsorbates on the lowest excited states of a methanethiol-CdSe conjugate

Bioconjugated CdSe quantum dots are promising reagents for bioimaging applications. Experimentally, the binding of a short peptide has been found to redshift the optical absorption of nanoclusters [J. Tsay et al., J. Phys. Chem. B 109, 1669 (2005)]. This study examines this issue by performing density functional theory (DFT) and time-dependent-DFT calculations to study the ground state and low-lying excited states of (CdSe)(6)[SCH(3)](-), a transition metal complex built by binding methanethiolate to a CdSe molecular cluster. Natural bond orbital results show that the redshift is caused by ligand-inorganic cluster orbital interaction. The highest occupied molecular orbital (HOMO) of (CdSe)(6) is dominated by selenium 4p orbitals; in contrast, the HOMO of (CdSe)(6)[SCH(3)](-) is dominated by sulfur 3p orbitals. This difference shows that [SCH(3)](-) binding effectively introduces filled sulfur orbitals above the selenium 4p orbitals of (CdSe)(6). The resulting smaller HOMO-LUMO gap of (CdSe)(6)[SCH(3)](-) indeed leads to redshifts in its excitation energies compared to (CdSe)(6). In contrast, binding of multiple NH(3) destabilizes cadmium 5p orbitals, which contribute significantly to the lowest unoccupied molecular orbital (LUMO) of (CdSe)(6), while leaving the selenium 4p orbitals near the HOMO relatively unaffected. This has the effect of widening the HOMO-LUMO gap of (CdSe)(6)6NH(3) compared to (CdSe)(6). As expected, the excitation energies of the passivated (CdSe)(6)6NH(3) are also blueshifted compared to (CdSe)(6). As far as NH(3) is a faithful representation of a surfactant, the results clearly illustrate the differences between the electronic effects of an alkylthiolate versus those of surfactant molecules. Surface passivation of (CdSe)(6)[SCH(3)](-) is then simulated by coating it with multiple NH(3) molecules. The results suggest that the [SCH(3)](-) adsorption induces a redshift in the excitation energies in a surfactant environment.

Toward Single-Molecule Optical Mapping of the Epigenome

The past decade has seen an explosive growth in the utilization of single-molecule techniques for the study of complex systems. The ability to resolve phenomena otherwise masked by ensemble averaging has made these approaches especially attractive for the study of biological systems, where stochastic events lead to inherent inhomogeneity at the population level. The complex composition of the genome has made it an ideal system to study at the single-molecule level, and methods aimed at resolving genetic information from long, individual, genomic DNA molecules have been in use for the last 30 years. These methods, and particularly optical-based mapping of DNA, have been instrumental in highlighting genomic variation and contributed significantly to the assembly of many genomes including the human genome. Nanotechnology and nanoscopy have been a strong driving force for advancing genomic mapping approaches, allowing both better manipulation of DNA on the nanoscale and enhanced optical resolving power for analysis of genomic information. During the past few years, these developments have been adopted also for epigenetic studies. The common principle for these studies is the use of advanced optical microscopy for the detection of fluorescently labeled epigenetic marks on long, extended DNA molecules. Here we will discuss recent single-molecule studies for the mapping of chromatin composition and epigenetic DNA modifications, such as DNA methylation.

Effects of bioconjugation on the structures and electronic spectra of CdSe: density functional theory study of CdSe-adenine complexes.

We present density functional theory (DFT) and time-dependent DFT (TD-DFT) study of the structures and electronic spectra of small CdSe nanocluster-adenine complexes Cd(n)Se(n)-adenine (n = 3, 6, 10, 13). We examine the changes in the geometries and excitation spectra of the nanoclusters induced by DNA base-binding. By comparing the results calculated for the bare (Cd(n)Se(n)), hydrogen-passivated (Cd(n)Se(n)H(2n)), as well as the corresponding adenine (Ade)-bound clusters (Cd(n)Se(n)-Ade, Cd(n)Se(n)H(2n)-Ade, Cd(n)Se(n)H(2n-2)-Ade), we find that binding with Ade slightly blue-shifts (up to 0.18 eV) the electronic excitations of bare nanoclusters but strongly red-shifts (<1.2 eV) those of hydrogen-passivated nanoclusters. Natural bond orbital analysis shows that the LUMO of Cd(n)Se(n)H(2n)-Ade is a pi* orbital located on the purine ring.

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase

Significance Transcription initiation by RNA polymerase (RNAP) is a highly regulated rate-limiting step in many genes and involves numerous intermediate states that remain incompletely understood. Here, we report the characterization of a previously hypothesized slow initiation pathway involving RNAP backtracking and pausing. This backtracked and paused state is observed when all nucleoside triphosphates (NTPs) are present at physiologically relevant concentrations, but becomes more prevalent with unbalanced NTP levels, which may occur in vivo under conditions of metabolic stress. Pausing and backtracking in initiation may play an important role in regulating RNAP transcription. Moreover, similar RNA backtracked states may contribute to promoter-proximal pausing among eukaryotic RNA polymerase II enzymes. Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP–GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused–backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes.

FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET

Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete software package for smFRET burst analysis is freely available to date. In this paper, we introduce FRETBursts, an open source software for analysis of freely-diffusing smFRET data. FRETBursts allows executing all the fundamental steps of smFRET bursts analysis using state-of-the-art as well as novel techniques, while providing an open, robust and well-documented implementation. Therefore, FRETBursts represents an ideal platform for comparison and development of new methods in burst analysis. We employ modern software engineering principles in order to minimize bugs and facilitate long-term maintainability. Furthermore, we place a strong focus on reproducibility by relying on Jupyter notebooks for FRETBursts execution. Notebooks are executable documents capturing all the steps of the analysis (including data files, input parameters, and results) and can be easily shared to replicate complete smFRET analyzes. Notebooks allow beginners to execute complex workflows and advanced users to customize the analysis for their own needs. By bundling analysis description, code and results in a single document, FRETBursts allows to seamless share analysis workflows and results, encourages reproducibility and facilitates collaboration among researchers in the single-molecule community.

Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Watching single molecules in motion Structural techniques such as x-ray crystallography and electron microscopy give insight into how macromolecules function by providing snapshots of different conformational states. Function also depends on the path between those states, but to see that path involves watching single molecules move. This became possible with the advent of single-molecule Förster resonance energy transfer (smFRET), which was first implemented in 1996. Lerner et al. review how smFRET has been used to study macromolecules in action, providing mechanistic insights into processes such as DNA repair, transcription, and translation. They also describe current limitations of the approach and suggest how future developments may expand the applications of smFRET. Science, this issue p. eaan1133 BACKGROUND Biomolecular mechanisms are typically inferred from static structural “snapshots” obtained by x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo–electron microscopy (cryo-EM). In these approaches, mechanisms have to be validated using additional information from established biochemical and biophysical assays. However, linking conformational states to biochemical function requires the ability to resolve structural dynamics, as macromolecular structure can be intrinsically dynamic or altered upon ligand binding. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying such structural dynamics under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments both confirmed previous hypotheses and discovered new fundamental biological mechanisms relevant for DNA maintenance, replication and transcription, translation, protein folding, enzymatic function, and membrane transport. We review the evolution of smFRET as a key tool for “dynamic structural biology” over the past 22 years and highlight the prospects for its use in applications such as biosensing, high-throughput screening, and molecular diagnostics. ADVANCES FRET was first identified in the 1920s by Cario, Franck, and Perrin. In the late 1940s, Förster and Oppenheimer independently formulated a quantitative theory of the energy transfer between a pair of point dipoles. Stryer and Haugland verified this theory in the late 1960s and coined the term “spectroscopic ruler” for FRET. Simultaneously, Hirschfeld, and later Moerner and Orrit, pioneered optical single-molecule detection methods leading to the first demonstration of smFRET in 1996. This breakthrough made it possible to study heterogeneous systems, dynamic processes, and transient conformational changes on the nanometer scale. The smFRET technique was rapidly adopted by various research groups to provide mechanistic answers in diverse areas of biological research. In early pioneering applications of smFRET in biochemistry, Ha et al. visualized the conformational dynamics of the staphylococcal nuclease enzyme; Deniz et al. obtained information on the structural dynamics of double-stranded DNA; and Zhuang et al. studied the conformation of individual RNA enzyme molecules and their folding dynamics in equilibrium. These pioneering studies were followed by others that used smFRET to unravel the inner workings of helicases and topoisomerases, DNA replication, DNA repair, transcription, translation, enzymatic reactions, molecular motors, membrane proteins, nucleic acids, protein and RNA folding, ribozyme catalysis, and many other molecular mechanisms. OUTLOOK During the past two decades, smFRET has grown into a mature toolset with capabilities to explore dynamic structural biology for both equilibrium and non-equilibrium reactions. The one-dimensional (“ruler”) character of the FRET approach, however, only captures the complex three-dimensional structure of a system and needs to be complemented by other techniques that can provide additional information about the respective biochemical states of macromolecules. Approaches that explore smFRET combinations with other biophysical techniques (patch-clamp, optical, and magnetic tweezers; atomic force microscopy; microfluidics) or photophysical effects are hence gaining attention. Although smFRET is particularly useful for the observation of dynamic conformational changes and subpopulations, FRET efficiencies also carry very precise information on the actual distance between fluorophores attached to distinct moieties of a macromolecule. As shown by recent work from many laboratories (such as those of Seidel, Michaelis, Hugel, and Grubmüller), this quantitative information can be used to help define biological structures and in the future should find a place in the protein database of molecular structures. smFRET has so far mostly been used for in vitro experiments but can be used additionally to monitor conformational dynamics and heterogeneity in live cells. “In vivo smFRET” has recently emerged as a promising methodology, demonstrated by the groups of Sakon, Weninger, Schuler, and Kapanidis among others. We envision that further technological developments will expand smFRET applications beyond dynamic structural biology to allow fast nonequilibrium kinetic studies, high-throughput drug screening, and molecular diagnostics. Advancements of these applications will be impactful for systems that are highly heterogeneous and dynamic. Dynamic structural biology using smFRET. Left: Principle of FRET as a molecular ruler. In a system with a pair of dyes, after the donor dye (D) is excited, it transfers the excitation energy to a nearby acceptor dye (A; top) with an efficiency (E) that depends on the sixth power of the distance between the dyes (bottom). Right: Use of FRET to study structural dynamics at the single-macromolecule level. The experimental setup (top), a combination of single-molecule fluorescence microscopy and spectroscopy, can be used to determine conformational states or dynamics in solution or on immobilized molecules. Here E is calculated per each single-molecule burst of photons, and bursts (n) are accumulated in E histograms (middle) or for different time bins to form a single-molecule E trajectory (bottom). Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

Multispot single-molecule FRET: High-throughput analysis of freely diffusing molecules

We describe an 8-spot confocal setup for high-throughput smFRET assays and illustrate its performance with two characteristic experiments. First, measurements on a series of freely diffusing doubly-labeled dsDNA samples allow us to demonstrate that data acquired in multiple spots in parallel can be properly corrected and result in measured sample characteristics consistent with those obtained with a standard single-spot setup. We then take advantage of the higher throughput provided by parallel acquisition to address an outstanding question about the kinetics of the initial steps of bacterial RNA transcription. Our real-time kinetic analysis of promoter escape by bacterial RNA polymerase confirms results obtained by a more indirect route, shedding additional light on the initial steps of transcription. Finally, we discuss the advantages of our multispot setup, while pointing potential limitations of the current single laser excitation design, as well as analysis challenges and their solutions.