Transcription Increases the Cooperativity of Ribonucleoprotein Assembly.

The synthesis of new ribosomes begins during transcription of the rRNA and is widely assumed to follow an orderly 5' to 3' gradient. To visualize co-transcriptional assembly of ribosomal protein-RNA complexes in real time, we developed a single-molecule platform that simultaneously monitors transcription and protein association with the elongating transcript. Unexpectedly, the early assembly protein uS4 binds newly made pre-16S rRNA only transiently, likely due to non-native folding of the rRNA during transcription. Stable uS4 binding became more probable only in the presence of additional ribosomal proteins that bind upstream and downstream of protein uS4 by allowing productive assembly intermediates to form earlier. We propose that dynamic sampling of elongating RNA by multiple proteins overcomes heterogeneous RNA folding, preventing assembly bottlenecks and initiating assembly within the transcription time window. This may be a common feature of transcription-coupled RNP assembly.

Recycling of bacterial RNA polymerase by the Swi2/Snf2 ATPase RapA.

Free-living bacteria have regulatory systems that can quickly reprogram gene transcription in response to changes in the cellular environment. The RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, may facilitate such reprogramming, but the mechanisms by which it does so are unclear. We used multiwavelength single-molecule fluorescence microscopy in vitro to examine RapA function in the Escherichia coli transcription cycle. In our experiments, RapA at <5 nM concentration did not appear to alter transcription initiation, elongation, or intrinsic termination. Instead, we directly observed a single RapA molecule bind specifically to the kinetically stable post termination complex (PTC)-consisting of core RNA polymerase (RNAP)-bound sequence nonspecifically to double-stranded DNA-and efficiently remove RNAP from DNA within seconds in an ATP-hydrolysis-dependent reaction. Kinetic analysis elucidates the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. This study defines how RapA participates in the transcription cycle between termination and initiation and suggests that RapA helps set the balance between global RNAP recycling and local transcription reinitiation in proteobacterial genomes.

Driving forces of the complex formation between highly charged disordered proteins.

Highly disordered complexes between oppositely charged intrinsically disordered proteins present a new paradigm of biomolecular interactions. Here, we investigate the driving forces of such interactions for the example of the highly positively charged linker histone H1 and its highly negatively charged chaperone, prothymosin α (ProTα). Temperature-dependent single-molecule Förster resonance energy transfer (FRET) experiments and isothermal titration calorimetry reveal ProTα-H1 binding to be enthalpically unfavorable, and salt-dependent affinity measurements suggest counterion release entropy to be an important thermodynamic driving force. Using single-molecule FRET, we also identify ternary complexes between ProTα and H1 in addition to the heterodimer at equilibrium and show how they contribute to the thermodynamics observed in ensemble experiments. Finally, we explain the observed thermodynamics quantitatively with a mean-field polyelectrolyte theory that treats counterion release explicitly. ProTα-H1 complex formation resembles the interactions between synthetic polyelectrolytes, and the underlying principles are likely to be of broad relevance for interactions between charged biomolecules in general.

Single-Molecule Spectroscopy and Super-Resolution Mapping of Physicochemical Parameters in Living Cells.

By superlocalizing the positions of millions of single molecules over many camera frames, a class of super-resolution fluorescence microscopy methods known as single-molecule localization microscopy (SMLM) has revolutionized how we understand subcellular structures over the past decade. In this review, we highlight emerging studies that transcend the outstanding structural (shape) information offered by SMLM to extract and map physicochemical parameters in living mammalian cells at single-molecule and super-resolution levels. By encoding/decoding high-dimensional information-such as emission and excitation spectra, motion, polarization, fluorescence lifetime, and beyond-for every molecule, and mass accumulating these measurements for millions of molecules, such multidimensional and multifunctional super-resolution approaches open new windows into intracellular architectures and dynamics, as well as their underlying biophysical rules, far beyond the diffraction limit.

Single-molecule fluorescence imaging and deep learning reveal highly heterogeneous aggregation of amyloid-ß 42.

Polymorphism in the structure of amyloid fibrils suggests the existence of many different assembly pathways. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity, but in practice it is extremely difficult to probe individual aggregation pathways in a mixture. Here, we present development of a method combining single-molecule fluorescence lifetime imaging and deep learning for monitoring individual fibril formation in real time and their high-throughput analysis. A deep neural network (FNet) separates an image of highly overlapping fibrils into single fibril images, which allows for tracking the growth and changes in characteristics of individual fibrils. Using this method, we investigated aggregation of the 42-residue amyloid-ß peptide (Aß42). We demonstrate that highly heterogeneous fibril formation can be quantitatively characterized in terms of the number of cross-ß subunits, elongation speed, growth polarity, and conformation of fibrils. Tracking individual fibril formation and growth also leads to the discovery of a general nucleation mechanism (termed heterogeneous secondary nucleation), where a fibril is formed on the surface of an oligomer with a different structure. Our development will be broadly applicable to characterization of heterogeneous aggregation processes of other proteins.

ApcE plays an important role in light-induced excitation energy dissipation in the Synechocystis PCC6803 phycobilisomes.

Phycobilisomes (PBs) play an important role in cyanobacterial photosynthesis. They capture light and transfer excitation energy to the photosynthetic reaction centres. PBs are also central to some photoprotective and photoregulatory mechanisms that help sustain photosynthesis under non-optimal conditions. Amongst the mechanisms involved in excitation energy dissipation that are activated in response to excessive illumination is a recently discovered light-induced mechanism that is intrinsic to PBs and has been the least studied. Here, we used single-molecule spectroscopy and developed robust data analysis methods to explore the role of a terminal emitter subunit, ApcE, in this intrinsic, light-induced mechanism. We isolated the PBs from WT Synechocystis PCC 6803 as well as from the ApcE-C190S mutant of this strain and compared the dynamics of their fluorescence emission. PBs isolated from the mutant (i.e., ApcE-C190S-PBs), despite not binding some of the red-shifted pigments in the complex, showed similar global emission dynamics to WT-PBs. However, a detailed analysis of dynamics in the core revealed that the ApcE-C190S-PBs are less likely than WT-PBs to enter quenched states under illumination but still fully capable of doing so. This result points to an important but not exclusive role of the ApcE pigments in the light-induced intrinsic excitation energy dissipation mechanism in PBs.

Deuterium Isotope Effect in Single Molecule Photophysics and Photochemistry of Hypericin.

The peripherical protons of the dye molecule hypericin can undergo structural interconversion (tautomerization) between different isomers separated by a low energy barrier with rates that depends sensitively on the interaction with local chemical environment defined by the nature of host material. We investigate the deuterium (D) isotope effect of hypericin tautomerism at the single-molecule level to avoid ensemble averaging in different polymer matrices by a combined spectroscopic and computational approach. In the 'innocent' PMMA matrix only intramolecular isotope effects on the internal conversion channel and tautomerization are observed; while PVA specifically interacts with the probe via H- and D-bonding. This establishes a single molecular picture on intra- and intermolecular nano-environment effects to control chromophore photophysics and -chemistry.

Hexylation Stabilises Twisted Backbone Configurations in the Prototypical Low-Bandgap Copolymer PCDTBT.

Conjugated donor-acceptor copolymers hold great potential as materials for high-performance organic photovoltaics, organic transistors and organic thermoelectric devices. Their low optical bandgap is achieved by alternation of donor and acceptor moieties along the polymer chain, leading to a pronounced charge-transfer character of electronic excitations. However, the influence of appended side chains and of chemical defects of the backbone on their photophysical and conformational properties remains largely unexplored on the level of individual chains. Here, we employ room temperature single-molecule photoluminescence spectroscopy on four compounds based on the prototypical copolymer PCDTBT with systematically changed chemical structure. Our results show that an increasing density of statistically added hexyl chains to the TBT comonomer distorts the molecular conformation, likely through the increase of average dihedral angles along the backbone. We find that, although the conformation becomes more twisted with high hexyl density, the side chains appear to stabilize the backbone in this twisted conformation. In addition, we demonstrate that homocoupling defects along the backbone barely influence the PL spectra of single chains, and thus intra-chain electronic properties.

Localization Study of Photostable Alexa 488 at Single Molecule Level.

Understanding the relationships between molecular organization and dynamics of a complex system is very important to understand the photophysical properties of such system. This paper focuses on a novel strategy based on single molecule spectroscopy and single molecule localization microscopy to elucidate the photostability and localization of a fluorophore molecule on a 2D biomembrane. Improvement of in-plane resolution of a signal in a nano-dimension within the diffraction limit has been discussed in a new way. And, how this better in-plane resolution information can be used for precise localization of a single molecule on a 2D system has also been discussed.

Single-Molecule Continuous-Wave Terahertz Rectification Spectroscopy and Microscopy.

We report rectification spectroscopy (RS) for single molecules performed with continuous-wave terahertz (CW THz) radiation at the tunneling junction of a scanning tunneling microscope (STM) at 8 K. CW THz-RS serves as a new technique in single-molecule vibrational and magnetic excitation spectroscopy besides inelastic electron tunneling spectroscopy (IETS). By quantitatively studying IETS and THz RS, we show that CW THz induces a sinusoidal bias modulation with amplitude linearly dependent on the THz far-field amplitude. Such THz-induced bias modulation amplitude appears to be sensitive to the THz beam alignment but insensitive to variation in the tunneling gap far smaller than the THz wavelength.

Multicolor 3D Orbital Tracking.

Feedback-based single-particle tracking (SPT) is a powerful technique for investigating particle behavior with very high spatiotemporal resolution. The ability to follow different species and their interactions independently adds a new dimension to the information available from SPT. However, only a few approaches have been expanded to multiple colors and no method is currently available that can follow two differently labeled biomolecules in 4 dimensions independently. In this proof-of-concept paper, the new modalities available when performing 3D orbital tracking with a second detection channel are demonstrated. First, dual-color tracking experiments are described studying independently diffusing particles of different types. For interacting particles where their motion is correlated, a second modality is implemented where a particle is tracked in one channel and the position of the second fluorescence species is monitored in the other channel. As a third modality, 3D orbital tracking is performed in one channel while monitoring its spectral signature in a second channel. This last modality is used to successfully readout accurate Förster Resonance Energy Transfer (FRET) values over time while tracking a mobile particle.

Depletion interactions modulate the binding between disordered proteins in crowded environments.

Intrinsically disordered proteins (IDPs) abound in cellular regulation. Their interactions are often transitory and highly sensitive to salt concentration and posttranslational modifications. However, little is known about the effect of macromolecular crowding on the interactions of IDPs with their cellular targets. Here, we investigate the influence of crowding on the interaction between two IDPs that fold upon binding, with polyethylene glycol as a crowding agent. Single-molecule spectroscopy allows us to quantify the effects of crowding on a comprehensive set of observables simultaneously: the equilibrium stability of the complex, the association and dissociation kinetics, and the microviscosity, which governs translational diffusion. We show that a quantitative and coherent explanation of all observables is possible within the framework of depletion interactions if the polymeric nature of IDPs and crowders is incorporated based on recent theoretical developments. The resulting integrated framework can also rationalize important functional consequences, for example, that the interaction between the two IDPs is less enhanced by crowding than expected for folded proteins of the same size.

Charge Transfer-Mediated Dramatic Enhancement of Raman Scattering upon Molecular Point Contact Formation.

Charge-transfer enhancement of Raman scattering plays a crucial role in current-carrying molecular junctions. However, the microscopic mechanism of light scattering in such nonequilibrium systems is still imperfectly understood. Here, using low-temperature tip-enhanced Raman spectroscopy (TERS), we investigate how Raman scattering evolves as a function of the gap distance in the single C60-molecule junction consisting of an Ag tip and various metal surfaces. Precise gap-distance control allows the examination of two distinct transport regimes, namely tunneling regime and molecular point contact (MPC). Simultaneous measurement of TERS and the electric current in scanning tunneling microscopy shows that the MPC formation results in dramatic Raman enhancement that enables one to observe the vibrations undetectable in the tunneling regime. This enhancement is found to commonly occur not only for coinage but also transition metal substrates. We suggest that the characteristic enhancement upon the MPC formation is rationalized by charge-transfer excitation.

Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities.

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation.

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts, and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals.

Real-Time Feedback-Driven Single-Particle Tracking: A Survey and Perspective.

Real-time feedback-driven single-particle tracking (RT-FD-SPT) is a class of techniques in the field of single-particle tracking that uses feedback control to keep a particle of interest in a detection volume. These methods provide high spatiotemporal resolution on particle dynamics and allow for concurrent spectroscopic measurements. This review article begins with a survey of existing techniques and of applications where RT-FD-SPT has played an important role. Each of the core components of RT-FD-SPT are systematically discussed in order to develop an understanding of the trade-offs that must be made in algorithm design and to create a clear picture of the important differences, advantages, and drawbacks of existing approaches. These components are feedback tracking and control, ranging from simple proportional-integral-derivative control to advanced nonlinear techniques, estimation to determine particle location from the measured data, including both online and offline algorithms, and techniques for calibrating and characterizing different RT-FD-SPT methods. Then a collection of metrics for RT-FD-SPT is introduced to help guide experimentalists in selecting a method for their particular application and to help reveal where there are gaps in the techniques that represent opportunities for further development. Finally, this review is concluded with a discussion on future perspectives in the field.

Reverse Intersystem Crossing of Single Deuterated Perylene Molecules in a Dibenzothiophene Matrix.

Intersystem crossing to the long-lived metastable triplet state is often a strong limitation on fluorescence brightness of single molecules, particularly for perylene in various matrices. In this paper, we report on a strong excitation-induced reverse intersystem crossing (rISC), a process where single perylene molecules in a dibenzothiophene matrix recover faster from the triplet state, turning into bright emitters at saturated excitation powers. With a detailed study of single-molecule fluorescence autocorrelations, we quantify the effect of rISC. The intrinsic lifetimes found for the two effective triplet states (8.5±0.4 ms and 64±12 ms) become significantly shorter, into the sub-millisecond range, as the excitation power increases and fluorescence brightness is ultimately enhanced at least fourfold. Our results are relevant for the understanding of triplet state manipulation of single-molecule quantum emitters and for markedly improving their brightness.

Reaction Cycle of Operating Pump Protein Studied with Single-Molecule Spectroscopy.

Biological machinery relies on nonequilibrium dynamics to maintain stable directional fluxes through complex reaction cycles. For such reaction cycles, the presence of microscopically irreversible conformational transitions of the protein, and the accompanying entropy production, is of central interest. In this work, we use multidimensional single-molecule fluorescence lifetime correlation spectroscopy to measure the forward and reverse conformational transitions of bacteriorhodopsin during trans-membrane H+ pumping. We quantify the flux, affinity, enthalpy and entropy production through portions of the reaction cycle as a function of temperature. We find that affinity of irreversible conformational transitions decreases with increasing temperature, resulting in diminishing flux and entropy production. We show that the temperature dependence of the transition affinity is well fit by the Gibbs-Helmholtz relation, allowing the ΔHtrans to be experimentally extracted.

Integrating single-molecule spectroscopy and simulations for the study of intrinsically disordered proteins.

Over the last two decades, intrinsically disordered proteins and protein regions (IDRs) have emerged from a niche corner of biophysics to be recognized as essential drivers of cellular function. Various techniques have provided fundamental insight into the function and dysfunction of IDRs. Among these techniques, single-molecule fluorescence spectroscopy and molecular simulations have played a major role in shaping our modern understanding of the sequence-encoded conformational behavior of disordered proteins. While both techniques are frequently used in isolation, when combined they offer synergistic and complementary information that can help uncover complex molecular details. Here we offer an overview of single-molecule fluorescence spectroscopy and molecular simulations in the context of studying disordered proteins. We discuss the various means in which simulations and single-molecule spectroscopy can be integrated, and consider a number of studies in which this integration has uncovered biological and biophysical mechanisms.

Resolving the Triexciton Recombination Pathway in CdSe/CdS Nanocrystals through State-Specific Correlation Measurements.

As luminescence applications of colloidal semiconductor nanocrystals push toward higher excitation flux conditions, there is an increased need to both understand and potentially control emission from multiexciton states. We develop a spectrally resolved correlation method to study the triply excited state that enables direct measurements of the recombination pathway for the triexciton, rather than relying on indirect extraction of rates. We demonstrate that, for core-shell CdSe-CdS nanocrystals, triexciton emission arises exclusively from the band-edge S-like state. Time-dependent density functional theory and extended particle-in-a-sphere calculations demonstrate that reduced carrier overlap induced by the core-shell heterostructure can account for the lack of emission observed from the P-like state. These results provide a potential avenue for the control of nanocrystal luminescence, where core-shell heterostructures can be leveraged to control carrier separation and therefore maintain emission color purity over a broader range of excitation fluxes.