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We have developed a high-throughput, full field-of-view (FOV) spectroscopic single-molecule fluorescence instrument, overcoming distortion issues with gratings and prisms. Our calibration techniques ensure spectral uniformity across the whole FOV, and our two-step Gaussian fit method enables better than 2 nm precision in spectral peak determination for accurate molecule identification, making it a valuable tool for multi-color single-molecule detection and analysis.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1’s essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1’s conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

Controlling the light emitted by individual molecules is instrumental to a number of advanced nanotechnologies ranging from super-resolution bioimaging and molecular sensing to quantum nanophotonics. Molecular emission can be tailored by modifying the local photonic environment, for example, by precisely placing a single molecule inside a plasmonic nanocavity with the help of DNA origami. Here, using this scalable approach, we show that commercial fluorophores may experience giant Purcell factors and Lamb shifts, reaching values on par with those recently reported in scanning tip experiments. Engineering of plasmonic modes enables cavity-mediated fluorescence far detuned from the zero-phonon-line (ZPL)─at detunings that are up to 2 orders of magnitude larger than the fluorescence line width of the bare emitter and reach into the near-infrared. Our results point toward a regime where the emission line width can become dominated by the excited-state lifetime, as required for indistinguishable photon emission, bearing relevance to the development of nanoscale, ultrafast quantum light sources and to the quest toward single-molecule cavity QED. In the future, this approach may also allow the design of efficient quantum emitters at infrared wavelengths, where standard organic sources have a reduced performance.

Cyanine dyes are exceptionally useful probes for a range of fluorescence‐based applications, but their photon output can be limited by trans‐to‐cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA‐nanoantenna, in single‐molecule Förster‐resonance energy transfer (FRET) applications, far‐red fluorescence‐lifetime imaging microscopy (FLIM), and single‐molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance. [ABSTRACT FROM AUTHOR]

Cyanobacterial photosystem I (PSI) constitutes monomeric and trimeric pigment–protein complexes whose optical properties are marked by the presence of long-wavelength absorption bands. In spite of numerous experimental studies, the nature of these bands is still under debate and requires intensive theoretical analysis. Collecting together the data of linear spectroscopy and single-molecule spectroscopy (SMS) of PSI from Arthrospira platensis, we performed quantum modeling of the optical response based on molecular exciton theory (ET) and the multimode Brownian oscillator model (MBOM). Applying MBOM, the spectra of the red antenna state were calculated considering a particular for each red state adjustment of the low-frequency vibronic modes. Within the framework of our PSI exciton model it was shown that the coupling energy between antenna chlorophylls cannot be a factor of the red states formation, thus the long-wavelength bands are calculated without attribution to so-called antenna red chlorophylls. By the fitting of Huang–Rhys factors and frequencies for the lowest vibronic modes, we were able to reproduce the effects of strong and weak electron–phonon coupling experimentally observed in SMS spectra of red antenna states. Based on our theoretical calculations and also analysis of existing crystal structures of cyanobacterial PSI, we assumed that long-wavelength Chls can be localized in the peripheral protein subunits containing one or two pigment molecules. [ABSTRACT FROM AUTHOR]

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 mu s to 1 ms. Distance distributions caused by fluctuations faster than 100 mu s are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (l) to the -1/2 power (l(-1/2)), and that the average contribution to the contour length per base is 0.40-0.45 nm. We study unfolded chymotrypsin inhibitor 2 (02) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded C12 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.

We use alternating-laser excitation to achieve fluorescence-aided molecule sorting (FAMS) and enable simultaneous analysis of bionnolecular structure and interactions at the level of single molecules. This was performed by labeling biomolecules with fluorophores that serve as donor-acceptor pairs for Forster resonance energy transfer, and by using alternating-laser excitation to excite directly both donors and acceptors present in single diffusing molecules. Emissions were reduced to the distance-dependent ratio E, and a distance-independent, stoichiometry-based ratio S. Histograms of E and S sorted species based on the conformation and association status of each species. S was sensitive to the stoichiometry and relative brightness of fluorophores in single molecules, observables that can monitor oligomerization and local-environment changes, respectively. FAMS permits equilibrium and kinetic analysis of macromolecule-ligand interactions; this was validated by measuring equilibrium and kinetic dissociation constants for the interaction of Escherichia coli catabolite activator protein with DNA. FAMS is a general platform for ratiometric measurements that report on structure, dynamics, stoichiometries, environment, and interactions of diffusing or immobilized molecules, thus enabling detailed mechanistic studies and ultrasensitive diagnostics.

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis., Competing Interests: TP, CH, RB, MD, CL, AV, JF, CH, SI, TV, BF, HG, JK, AS, CS, CH No competing interests declared, (© 2023, Peulen, Hengstenberg et al.)

How the eukaryotic 43S preinitiation complex scans along the 5′ untranslated region (5′ UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5′ UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5′ direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5′ UTR structures and upstream near-cognate start codons. [Display omitted] • Real-time tracking of 43S-mRNA binding, scanning, and 60S joining • Scanning occurs at ∼100 nucleotides per second • Start codon-proximal 5′ UTR hairpin sequences induce scanning direction fluctuations • Downstream hairpin sequences promote near-cognate start codon initiation Direct observation of single initiating eukaryotic ribosomes establishes a quantitative framework of how ribosomes scan the mRNA 5′ UTR to locate the start codon and how this process is regulated by mRNA sequence and structure. [ABSTRACT FROM AUTHOR]

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCRtargeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence techniques are ideally suited to provide information about the structure-function-dynamics relationship of a biomolecule as static and dynamic heterogeneity can be easily detected. However, what type of single-molecule fluorescence technique is suited for which kind of biological question and what are the obstacles on the way to a successful single-molecule microscopy experiment? In this review, we provide practical insights into fluorescence-based single-molecule experiments aiming for scientists who wish to take their experiments to the single-molecule level. We especially focus on fluorescence resonance energy transfer (FRET) experiments as these are a widely employed tool for the investigation of biomolecular mechanisms. We will guide the reader through the most critical steps that determine the success and quality of diffusion-based confocal and immobilization-based total internal reflection fluorescence microscopy. We discuss the specific chemical and photophysical requirements that make fluorescent dyes suitable for single-molecule fluorescence experiments. Most importantly, we review recently emerged photoprotection systems as well as passivation and immobilization strategies that enable the observation of fluorescently labeled molecules under biocompatible conditions. Moreover, we discuss how the optical single-molecule toolkit has been extended in recent years to capture the physiological complexity of a cell making it even more relevant for biological research. [ABSTRACT FROM AUTHOR]

A principal limitation of single-molecule spectroscopy in solution is the diffusion-limited residence time of a given molecule within the detection volume. A common solution to this problem is to immobilize molecules of interest on a passivated glass surface for extending the observation time to obtain reliable data statistics. However, surface tethering of molecules often introduces artifacts, particularly when studying the structural dynamics of biomolecules. To circumvent this limitation, we investigated alternative ways to extend single-molecule observation times in solution without surface immobilization. Among various possibilities, the so-called anti-Brownian electro-kinetic trap (or ABEL trap) seems best suited to achieve this goal. The essential part of that trap is a feedback-controlled electro-kinetic steering of a molecule's position in reaction to its diffusive Brownian motion which is monitored by fluorescence, thus keeping the molecule within a sub-micron sized detection volume. Fluorescence trace recordings of over thousands of milliseconds duration on individual dye molecules within an ABEL trap have been reported. In this short review, we shall briefly discuss the principle and some results of ABEL trapping of individual molecules with possible extensions to future works. [ABSTRACT FROM AUTHOR]

The genetic information of every living organism is stored in its genomic DNA that is perceived as a chemically stable and robust macromolecule. But at the same time, to fulfil its functions properly, it also needs to be highly dynamic and flexible. This includes partial melting of the double helix or compaction and bending of the DNA often brought about by protein factors that are able to interact with DNA stretches in a specific and non-specific manner. The conformational changes in the DNA need to be understood in order to describe biological systems in detail. As these events play out on the nanometre scale, new biophysical approaches have been employed tomonitor conformational changes in this regime at the single-molecule level. Focusing on transcription factor action on promoter DNA, we discuss how current biophysical techniques are able to quantitatively describe this molecular process. [ABSTRACT FROM AUTHOR]

Abstract: Extremely pure polymer matrices were used for elucidating the fluorescence properties of singly isolated charge-transfer (CT) complexes formed between the donor N-ethylcarbazole and the acceptor 1,2,4,5-tetracyanobenzene. Simultaneous measurements (time traces of CT fluorescence intensities and lifetimes) using single-molecule fluorescence spectroscopy showed three patterns: (1) fluctuations in the fluorescence intensities and lifetimes seldom occurred, (2) the fluorescence intensities frequently fluctuated together with the lifetimes, or (3) in addition to the above fluctuations with time, blinking and/or off-states longer than 1s were observed. For methacrylate polymers, both the degree of fluctuations in the CT fluorescence lifetimes and the percentage of the CT complexes showing off-states increased with the free volume of the host polymers. These results suggest that the degree of fluctuations in the relative geometrical arrangements of the donor and acceptor molecules is related to the availability of space in the host polymer, and that the free volume provides the necessary space for formation of non-fluorescent donor–acceptor geometries of the CT complexes and/or temporal dissociation of the CT complexes. Survival times of the CT fluorescence were also closely related with the free volume of the host polymers. [Copyright &y& Elsevier]

The breast and ovarian cancer suppressor BRCA2 controls the enzyme RAD51 during homologous DNA recombination (HDR) to preserve genome stability. BRCA2 binds to RAD51 through 8 conserved BRC repeat motifs dispersed in an 1127-residue region (BRCA2[BRC1-8]). Here, we show that BRCA2[BRC1-8] exerts opposing effects on the binding of RAD51 to single-stranded (ss) versus double-stranded (ds) DNA substrates, enhancing strand exchange. BRCA2[BRC1-8] alters the electrophoretic mobility of RAD51 bound to an ssDNA substrate, accompanied by an increase in ssDNA-bound protein assemblies, revealed by electron microscopy. Single-molecule fluorescence spectroscopy shows that BRCA2[BRC1-8] promotes RAD51 loading onto ssDNA. In contrast, BRCA2[BRC1-8] has a different effect on RAD51 assembly on dsDNA; it suppresses and slows this process. When homologous ssDNA and dsDNA are both present. BRCA2[BRC1-8] stimulates strand exchange, with delayed RAD51 loading onto dsDNA accompanying the appearance of joint molecules representing re-combination products. Collectively, our findings suggest that BRCA2[BRC1-8] targets RAD51 to ssDNA while inhibiting dsDNA binding and that these contrasting activities together bolster one another to stimulate HDR. Our work provides fresh insight into the mechanism of HDR in humans, and its regulation by the BRCA2 tumor suppressor. [ABSTRACT FROM AUTHOR]

A more complete depiction of protein energy landscapes includes the identification of different function-related conformational states and the determination of the pathways connecting them. We used total internal reflection fluorescence (TIRF) imaging to investigate the conformational dynamics of the human A 2A adenosine receptor (A 2A AR), a class A G protein-coupled receptor (GPCR), at the single-molecule level. Slow, reversible conformational exchange was observed among three different fluorescence emission states populated for agonist-bound A 2A AR. Transitions among these states predominantly occurred in a specific order, and exchange between inactive and active-like conformations proceeded through an intermediate state. Models derived from molecular dynamics simulations with available A 2A AR structures rationalized the relative fluorescence emission intensities for the highest and lowest emission states but not the transition state. This suggests that the functionally critical intermediate state required to achieve activation is not currently visualized among available A 2A AR structures. [Display omitted] • Single-molecule fluorescence observes slow dynamic behavior of A 2A AR complexes • Three different fluorescence emission states observed for A 2A AR complexes with agonists • Reversible transitions among different states occurred in a sequential order • A functionally critical transition state not represented among available structures Wei et al. use single-molecule fluorescence to observe slow exchange among at least three conformations of the agonist-bound A 2A adenosine receptor. Transitions from inactive to active-like conformations are sequential and must proceed through an intermediate state not currently represented among available structures. [ABSTRACT FROM AUTHOR]

Oligomerization of the HIV-1 protein Rev on the Rev Response Element (RRE) regulates nuclear export of genomic viral RNA and partially spliced viral mRNAs encoding for structural proteins. Single-molecule fluorescence spectroscopy has been used to dissect the multistep assembly pathway of this essential ribonucleo-protein, revealing dynamic intermediates and the mechanism of assembly. Assembly is initiated by binding of Rev to a high-affinity site in stem-loop IIB of the RRE and proceeds rapidly by addition of single Rev monomers, facilitated by cooperative Rev-Rev interactions on the RRE. Dwell-time analysis of fluorescence trajectories recorded during individual Rev-RRE assembly reactions has revealed the microscopic rate constants for several of the Rev monomer binding and dissociation steps. The high-affinity binding of multiple Rev monomers to the RRE is achieved on a much faster timescale than reported in previous bulk kinetic studies of Rev-RRE association, indicating that oligomerization is an early step in complex assembly. [ABSTRACT FROM AUTHOR]

The ribosome is the macromolecular machine responsible for translating the genetic code into polypeptide chains. Despite impressive structural and kinetic studies of the translation process, a number of challenges remain with respect to understanding the dynamic properties of the translation apparatus. Single-molecule techniques hold the potential of characterizing the structural and mechanical properties of macromolecules during their functional cycles in real time. These techniques often necessitate the specific coupling of biologically active molecules to a surface. Here, we describe a procedure for such coupling of functionally active ribosomes that permits single-molecule studies of protein synthesis. Oxidation with NaIO4 at the 3′ end of 23S rRNA and subsequent reaction with a biotin hydrazide produces biotinylated 70S ribosomes, which can be immobilized on a streptavidin-coated surface. The surface-attached ribosomes are fully active in poly(U) translation in vitro, synthesizing poly(Phe) at a rate of 3–6 peptide bonds/s per active ribosome at 37°C. Specificity of binding of biotinylated ribosomes to a streptavidin-coated quartz surface was confirmed by observation of individual fluorescently labeled, biotinylated 70S ribosomes, using total internal reflection fluorescence microscopy. Functional interactions of the immobilized ribosomes with various components of the protein synthesis apparatus are shown by use of surface plasmon resonance. [ABSTRACT FROM AUTHOR]

Abstract: In recent years, single-molecule methods have enabled many innovative studies in the life sciences, which generated unprecedented insights into the workings of many macromolecular machineries. Single-molecule studies of bioinorganic systems have been limited, however, even though bioinorganic chemistry represents one of the frontiers in the life sciences. With the hope to stimulate more interest in applying existing and developing new single-molecule methods to address compelling bioinorganic problems, this review discusses a few single-molecule fluorescence approaches that have been or can be employed to study the functions and dynamics of metalloproteins. We focus on their principles, features and generality, possible further bioinorganic applications, and experimental challenges. The fluorescence quenching via energy transfer approach has been used to study the O2-binding of hemocyanin, the redox states of azurin, and the folding dynamics of cytochrome c at the single-molecule level. Possible future applications of this approach to single-molecule studies of metalloenzyme catalysis and metalloprotein folding are discussed. The fluorescence quenching via electron transfer approach can probe the subtle conformational dynamics of proteins, and its possible application to probe metalloprotein structural dynamics is discussed. More examples are presented in using single-molecule fluorescence resonance energy transfer to probe metallochaperone protein interactions and metalloregulator–DNA interactions on a single-molecule basis. [Copyright &y& Elsevier]

Alternating-laser excitation (ALEX) spectroscopy has recently been added to the single-molecule spectroscopy toolkit. ALEX monitors interaction and stoichiometry of biomolecules, reports on biomolecular structure by measuring accurate Förster resonance energy transfer (FRET) efficiencies, and allows sorting of subpopulations on the basis of stoichiometry and FRET. Here, we demonstrate that a simple combination of one continuous-wave donor-excitation laser and one directly modulated acceptor-excitation laser (Periodic Acceptor eXcitation) is sufficient to recapitulate the capabilities of ALEX while minimizing the cost and complexity associated with use of modulation techniques. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence spectroscopy, and particularly its Förster resonance energy transfer implementation (SM-FRET), provides the opportunity to resolve the stochastic conformational fluctuations undergone by individual protein molecules while they fold-unfold, bind to their partners, or carry out catalysis. Such information is key to resolve the microscopic pathways and mechanisms underlying such processes, and cannot be obtained from bulk experiments. To fully resolve protein conformational dynamics, SM-FRET experiments need to reach microsecond, and even sub-microsecond, time resolutions. The key to reach such resolution lies in increasing the efficiency at which photons emitted by a single molecule are collected and detected by the instrument (photon count rates). In this chapter, we describe basic procedures that an end user can follow to optimize the confocal microscope optics in order to maximize the photon count rates. We also discuss the use of photoprotection cocktails specifically designed to reduce fluorophore triplet buildup at high irradiance (the major cause of limiting photon emission rates) while improving the mid-term photostability of the fluorophores. Complementary strategies based on the data analysis are discussed in depth by other authors in Chap. 14 ., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

G-protein-coupled receptors (GPCRs) mediate a wide range of human physiological functions by transducing extracellular ligand binding events into intracellular responses. GPCRs can activate parallel, independent signaling pathways mediated by G proteins or β-arrestins. Whereas "balanced" agonists activate both pathways equally, "biased" agonists dominantly activate one pathway, which is of interest for designing GPCR-targeting drugs because it may mitigate undesirable side effects. Previous studies demonstrated that β-arrestin activation is associated with transmembrane helix VII (TM VII) of GPCRs. Here, single-molecule fluorescence spectroscopy with the β 2 -adrenergic receptor (β 2 AR) in the ligand-free state showed that TM VII spontaneously fluctuates between one inactive and one active-like conformation. The presence of the β-arrestin-biased agonist isoetharine prolongs the dwell time of TM VII in the active-like conformation compared with the balanced agonist formoterol, suggesting that ligands can induce signaling bias by modulating the kinetics of receptor conformational exchange. • TM VII of β 2 AR naturally fluctuates between inactive and active-like conformations • Agonists prolong the dwell time of the active-like conformation of TM VII • A β-arrestin-biased agonist is more stabilizing than a balanced agonist TM helix VII of β 2 AR mediates cellular signaling via the β-arrestin pathway. Using an in vitro single-molecule fluorescence system, Lamichhane et al. present evidence that a β-arrestin-biased agonist induces signaling bias by controlling the kinetics of exchange between inactive and active-like conformations of TM VII. [ABSTRACT FROM AUTHOR]

Watching the conformational wanderings of protein molecules during their search for their native structure is a holy grail for protein folding experimentalists. Such capability is essential to provide reality checks to the complex mechanistic heterogeneity that theory and molecular simulations predict. Single-molecule fluorescence resonance energy transfer (SM-FRET) is an attractive technique to meet that end, but its time resolution was insufficient for the microsecond motions of folding proteins. The temporal resolution of SM-FRET pivots on how quickly photons emitted by an individual molecule can be collected in sufficient numbers as to minimize statistical shot noise. Recent multipronged advances in that front have led us to gain first access to fast folding dynamics. Center stage is the implementation of photochemical strategies to quickly recover fluorophores from long-lasting dark states. Improvements in optics and novel procedures for data analysis in probabilistic terms are additional contributions. In parallel, noninvasive methods for fluorescent labeling and immobilization of proteins have also been implemented. Here we discuss all these exciting developments, providing experimental guidelines and procedural details for the implementation of fast SM-FRET experiments on proteins., (© 2016 Elsevier Inc. All rights reserved.)

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β2-adrenergic receptor (β2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

Highly π-conjugated perylenediimide (PDI) oligomers are promising low band gap organic materials for various applications in optoelectronics. In this work, individual fluorescence dynamics of ethynylene- and butadiynylene-bridged dimeric and trimeric PDIs (PEP, PBP, and PEPEP) were monitored and analyzed by single-molecule fluorescence spectroscopy to gain information on the degree of extension of π-conjugation through the acetylene bridge in PDI multichromophores. The simultaneous measurements of fluorescence intensity, lifetime, and spectrum indicate a sequential decrease in π-conjugation upon photobleaching of PDI monomer units. Furthermore, Huang-Rhys (HR) factors, S, are obtained to evaluate the degree of electronic coupling in view of π-conjugation and overall rigidity between the PDI units in PDI oligomers at the single-molecule level. In addition, butadiynylene-bridged dimeric PDI (PBP) reveals conformational heterogeneity due to the long butadiynylene linker. These results suggest a new way to control the photophysical properties of the PDI multichromophoric system by expansion of π-conjugation and modification with different linker groups.