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• Single-molecule fluoresence spectroscopy is a key approach for the study of IDRs. • Molecular simulations provide atomistic insight into IDR conformation and dynamics. • Despite their importance, both sets of techniques have a number of limitations. • When combined they offer complimentary insight with largely orthogonal weaknesses. 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. [ABSTRACT FROM AUTHOR]

Acquiring events massively from single-molecule force spectroscopy (SMFS) experiments, which is crucial for revealing important biophysical information, is usually not straightforward. A significant amount of human labor is usually required to identify events in the measured force spectrum during measuring or before performing further data analysis. This prevents the experiment from being done in a fully-automated manner or scaling with the throughput of the measuring setup. In this work, we attempt to tackle this problem with a deep learning approach. A deep neural network model is developed to infer the occurrence of the events using the data stream from the measuring setup. We demonstrated that the proposed method could achieve high accuracy with force spectrums of a variety of samples from both optical tweezers and AFMs by learning from user-given samples instead of complicated manual algorithm designing or parameter tuning. Furthermore, we found that the trained model can be used to perform event detection on datasets measured from a different optical tweezer setup, showing the potential of being leveraged in more complex deep learning schemes. • This work proposed a user-friendly approach to massively locate the events of interest from experimental SMFS data streams. • To our best knowledge, this work is the first attempt to adopt deep learning methods to locate events in SMFS experiments. • It is demonstrated that the proposed method could learn to locate events for a variety of samples from different instruments including optical tweezers and AFMs. • This method could help to achieve automated measuring on SMFS high-throughput platforms or other deep-learning based SMFS signal analysis. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence resonance energy transfer (smFRET) is used to investigate the movement of the γ-subunit within the H+-ATPsynthase from chloroplasts during proton transport-coupled ATP synthesis. The FRET donor fluorophore is attached to residue γC322 and the FRET acceptor is non-covalently bound to a non-catalytic nucleotide binding site. The labeled enzyme is integrated into liposomes and ATP synthesis is started by an acid-base transition. A custom-built confocal microscope with two spectral detection channels is used to measure smFRET with freely diffusing proteoliposomes. Analysis of the smFRET time traces reveals stepwise distance changes between the two fluorophores. During catalysis the γ-subunit interacts with equal probability with each αβ-pair. In ADP-inhibited enzymes, the γ-subunit interacts with only one specific αβ-pair. Comparison of the smFRET data with cryo-EM data (Hahn, Vonck, Mills, Meier, & Kühlbrandt, 2018, Science, 360, pp. 620-628) indicated that the transition from the ADP-inhibited state to the active state is accompanied by a large rotational movement of the γ-subunit. [ABSTRACT FROM AUTHOR]

The photophysical properties of chromophores react very sensitively upon changes of their electrostatic environment. This allows conformational fluctuations of a protein to be monitored by optical spectroscopy via the fluctuations of the spectral signatures of chromophores that are embedded in the protein's matrix. However, to be successful as an approach this requires that the structural fluctuations within the proteins would occur synchronously. This restriction can be overcome by studying the proteins on an individual basis. In this contribution, we illustrate this approach on the example of the peripheral light-harvesting complexes from photosynthetic purple bacteria that contain bacteriochlorophyll a as natural cofactors. [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence has the unique ability to quantify small oligomers and track conformational changes at a single-protein level. Here we tackled one of the most extreme protein behaviors, found recently in an inflammation pathway. Upon danger recognition in the cytosol, NLRP3 recruits its signaling adaptor, ASC. ASC start polymerizing in a prion-like manner and the system goes in “overdrive” by producing a single micron-sized “speck.” By precisely controlling protein expression levels in an in vitro translation system, we could trigger the polymerization of ASC and mimic formation of specks in the absence of inflammasome nucleators. We utilized single-molecule spectroscopy to fully characterize prion-like behaviors and self-propagation of ASC fibrils. We next used our controlled system to monitor the conformational changes of ASC upon fibrillation. Indeed, ASC consists of a PYD and CARD domains, separated by a flexible linker. Individually, both domains have been found to form fibrils, but the structure of the polymers formed by the full-length ASC proteins remains elusive. For the first time, using single-molecule Förster resonance energy transfer, we studied the relative positions of the CARD and PYD domains of full-length ASC. An unexpectedly large conformational change occurred upon ASC fibrillation, suggesting that the CARD domain folds back onto the PYD domain. However, contradicting current models, the “prion-like” conformer was not initiated by binding of ASC to the NLRP3 platform. Rather, using a new method, hybrid between Photon Counting Histogram and Number and Brightness analysis, we showed that NLRP3 forms hexamers with self-binding affinities around 300 nM. Overall our data suggest a new mechanism, where NLRP3 can initiate ASC polymerization simply by increasing the local concentration of ASC above a supercritical level. [ABSTRACT FROM AUTHOR]

The ability of cells to sense and respond to mechanical forces is crucial for a wide range of developmental and pathophysiological processes. The molecular mechanisms underlying cellular mechanotransduction, however, are largely unknown because suitable techniques to measure mechanical forces across individual molecules in cells have been missing. In this article, we highlight advances in the development of molecular force sensing techniques and discuss our recently expanded set of FRET-based tension sensors that allows the analysis of mechanical forces with piconewton sensitivity in cells. In addition, we provide a theoretical framework for the design of additional tension sensor modules with adjusted force sensitivity. [ABSTRACT FROM AUTHOR]

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli have an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations (100pM) to exclude aggregation, we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP) by single-pair fluorescence resonance energy transfer and fluorescence correlation spectroscopy. We find that GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. Accelerated folding is achieved by encapsulation of folding intermediate in the GroEL/ES cage, independent of repetitive cycles of protein binding and release from GroEL. Moreover, photoinduced electron transfer experiments provided direct physical evidence that the confining environment of the chaperonin restricts polypeptide chain dynamics. This effect is mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. EL(KKK2)/ES functions as a passive cage in which folding occurs at the slow spontaneous rate. Taken together our findings suggest that protein encapsulation can accelerate folding by entropically destabilizing folding intermediates, in strong support of an active chaperonin mechanism in the folding of some proteins. Accelerated folding is biologically significant as it adjusts folding rates relative to the speed of protein synthesis. [ABSTRACT FROM AUTHOR]

Abstract: Harnessing the new bioremediation and biotechnology applications offered by the dissimilatory metal-reducing bacteria, Shewanella oneidensis MR-1, requires a clear understanding of its transcription machinery, a pivotal component in maintaining vitality and in responding to various conditions, including starvation and environmental stress. Here, we have reconstituted the S. oneidensis RNA polymerase (RNAP) core in vivo by generating a co-overexpression construct that produces a long polycistronic mRNA encoding all of the core subunits (α, β, β′, and ω) and verified that this reconstituted core is capable of forming fully functional holoenzymes with the S. oneidensis σ factors σ70, σ38, σ32, and σ24. Further, to demonstrate the applications for this reconstituted core, we report the application of single-molecule fluorescence resonance energy transfer (smFRET) assays to monitor the mechanisms of transcription by the S. oneidensis σ70-RNAP holoenyzme. These results show that the reconstituted transcription machinery from S. oneidensis, like its Escherichia coli counterpart, “scrunches” the DNA into its active center during initial transcription, and that as the holoenzyme transitions into elongation, the release of σ70 is non-obligatory. [Copyright &y& Elsevier]

Abstract: Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.e. Alexa 647 labeled antibodies, can be used as efficient photoswitches. In combination with localization microscopy approaches the method is ideally suited to study the spatial organization of proteins in sub-cellular structures and membranes. The simplicity of the method lies in the fact that standard immunocytochemistry assays together with photoswitchable carbocyanine fluorophores and conventional total internal reflection fluorescence (TIRF) microscopy can be used to achieve a lateral resolution of ∼ 20nm. We demonstrate subdiffraction-resolution fluorescence imaging of intracellular F0F1-ATP synthase and cytochrome c oxidase in the inner membrane of mitochondria. Besides the high localization precision of individual proteins we demonstrate how quantitative data, i.e. the protein distribution in the membrane, can be derived and compared. [Copyright &y& Elsevier]

Abstract: Misfolding and aggregation of amyloid-β peptide (Aβ) are widely recognized as causative events in Alzheimer’s disease (AD). Contrary to earlier hypotheses, recent studies have identified soluble Aβ oligomers as the pathogenic agents and documented neurodegenerative effects from species as small as dimers and trimers. As such, detection and characterization of the earliest Aβ oligomers are paramount to understanding, preventing, and treating AD. We have exploited quantized photobleaching from individual dye-labeled Aβ peptides to characterize monomers and small oligomers tethered to the surface of functionalized coverslips. In this way, we have directly determined reproducible distributions of peptide species in various sample environments. Fresh samples (30pM peptide) at pH 7.4 consist primarily of monomers and dimers. Both acidic conditions (pH 5.8) and zinc coordination promote greater association, which is further increased in samples prepared from more concentrated stocks. Acid- or zinc-promoted association is largely prevented and/or reversed by addition of the β-sheet breaker peptide iAβ5 or the chelator clioquinol, respectively. These results are qualitatively consistent with bulk-solution studies of Aβ40 and demonstrate a powerful approach for definitively characterizing Aβ oligomers. The methodology utilized here holds promise for assessing aggregation promoters and inhibitors and investigating peptide association in its earliest stages. [Copyright &y& Elsevier]

Abstract: Single-molecule fluorescence resonance energy transfer (SMFRET) was used to study the interaction of a 25-nucleotide (nt) DNA aptamer with its binding target, vascular endothelial growth factor (VEGF). Conformational dynamics of the aptamer were studied in the absence of VEGF in order to characterize fluctuations in the unbound nucleic acid. SMFRET efficiency distributions showed that, while the aptamer favors a base-paired conformation, there are frequent conversions to higher energy conformations. Conversions to higher energy structures were also demonstrated to be dependent on the concentration of Mg2+-counterion by an overall broadening of the SMFRET efficiency distribution at lower Mg2+ concentration. Introduction of VEGF caused a distinct increase in the frequency of lower SMFRET efficiencies, indicating that favorable interaction of the DNA aptamer with its VEGF target directs aptamer structure towards a more open conformation. [Copyright &y& Elsevier]

Vascular endothelial (VE) cadherin is the surface glycoprotein cadherin specific to the endothelium that mediates cell–cell adhesion and plays a major role in the remodeling, gating, and maturation of vascular vessels. To investigate the contribution of individual VE-cadherins to endothelial cell–cell interactions and investigate whether different classical cadherins display different kinetics and micromechanical properties, we characterize the binding properties of VE-cadherin/VE-cadherin bonds at single-molecule resolution and in living human umbilical vein endothelial cells (HUVECs). Our single-molecule force spectroscopy measurements reveal that type II VE-cadherin molecules form bonds that are less prone to rupture and display a higher tensile strength than bonds formed by classical type I neuronal (N) cadherin and epithelial (E) cadherin. The equilibrium lifetime of the VE-cadherin/VE-cadherin bond is significantly longer than formed by N-cadherin/N-cadherin bonds and E-cadherin/E-cadherin bonds. These results indicate that VE-cadherins form bonds that have kinetics and mechanical properties that are significantly different from those formed by classical type I cadherins, properties that are particularly well adapted to the barrier and adhesive functions of VE-cadherin in endothelial cell–cell junctions. [Copyright &y& Elsevier]

Single-molecule fluorescence (Förster) resonance energy transfer (FRET) experiments were performed on surface-immobilized RNase H molecules as a function of the concentration of the chemical denaturant guanidinium chloride (GdmCl). For comparison, we measured ensemble FRET on RNase H solutions. The single-molecule approach allowed us to study FRET distributions of the subpopulation of unfolded molecules without interference from the folded population. The unfolded ensemble experienced a continuous shift of the FRET efficiency distribution with increasing concentration of GdmCl, indicating a heterogeneous population of expanding, unfolded polypeptide chains. We have analyzed the behavior of the unfolded state quantitatively with a model in which the unfolded state is described by a continuum of substates, with the free energy of each substate linearly coupled to its m-value, the proportionality coefficient between free energy and denaturant activity. By fitting this model to the data, we have derived energetic and structural parameters that describe the unfolded state ensemble. Specifically, we have found that the average size of the unfolded state increases from 23–38Å between 0 and 6M denaturant. Excellent agreement was achieved between the fitted model and our FRET measurements, and with previously published nuclear magnetic resonance and small-angle X-ray scattering data. [Copyright &y& Elsevier]

Abstract: Nucleosomes are the basic units of eukaryotic chromatin structure. By restricting factor access to regulatory DNA sequences, nucleosomes significantly impact genomic processes such as transcription, and various mechanisms to alter nucleosome structure to relieve this repression have evolved. Both nucleosomes and processes that alter them are inherently dynamic in nature. Thus, studies of dynamics will be necessary to truly understand these relief mechanisms. We describe here the characteristics of a novel fluorescence resonance energy transfer-based reporter that can clearly signal the formation of a canonical nucleosome structure and follow conformational and compositional changes in that structure, both at the ensemble-average (bulk) and at the single molecule level. Labeled nucleosomes behave conformationally and thermodynamically like typical nucleosomes; thus they are relevant reporters of nucleosome behavior. Nucleosomes and free DNA are readily distinguishable at the single-molecule level. Thus, these labeled nucleosomes are well suited to studies of dynamic changes in nucleosome structure including single-molecule dynamics. [Copyright &y& Elsevier]

The molecular basis of high cooperativity in multi-subunit proteins is still unknown in most cases. Oxygen binding by multi-subunit hemocyanins produces two intrinsic spectroscopic signals which are, however, either limited to the UV or are very weak. Here we demonstrate that fluorescence labels emitting in the visible can be used as sensors for cooperative oxygen binding of hemocyanins. Fluorescence resonance energy transfer to the oxygenated active sites quenches the emission of the labels by roughly 50% upon oxygenation of the protein. The labels give strong and photo-stable emission, allowing imaging of single hemocyanin molecules. Therefore, this study opens up a new perspective for investigating the molecular basis of cooperative oxygen binding at the single-molecule level. In addition, another novel application is provided by these labels, i.e., the investigation of the influence of effectors by recording simultaneously the binding of oxygen in the visible and of effectors in the UV. [Copyright &y& Elsevier]

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP · CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP · CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP · CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP · CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP · CaM-DA molecules. The extension of this method to other biomolecules is also proposed. [Copyright &y& Elsevier]