Single-molecule Studies Research Articles

Enzyme Engineering by Force: DNA Springs for the Modulation of Biocatalytic Trajectories.

The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

Translational T-box riboswitches bind tRNA by modulating conformational flexibility

T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Hexagonal Plasmonic Arrays for High-Throughput Multicolor Single-Molecule Studies.

Nanophotonic biosensors offer exceptional sensitivity in the presence of strong background signals by enhancing and confining light in subwavelength volumes. In the field of nanophotonic biosensors, antenna-in-box (AiB) designs consisting of a nanoantenna within a nanoaperture have demonstrated remarkable single-molecule fluorescence detection sensitivities under physiologically relevant conditions. However, their full potential has not yet been exploited as current designs prohibit insightful correlative multicolor single-molecule studies and are limited in terms of throughput. Here, we overcome these constraints by introducing aluminum-based hexagonal close-packed AiB (HCP-AiB) arrays. Our approach enables the parallel readout of over 1000 HCP-AiBs with multicolor single-molecule sensitivity up to micromolar concentrations using an alternating three-color excitation scheme and epi-fluorescence detection. Notably, the high-density HCP-AiB arrays not only enable high-throughput studies at micromolar concentrations but also offer high single-molecule detection probabilities in the nanomolar range. We demonstrate that robust and alignment-free correlative multicolor studies are possible using optical fiducial markers even when imaging in the low millisecond range. These advancements pave the way for the use of HCP-AiB arrays as biosensor architectures for high-throughput multicolor studies on single-molecule dynamics.

Characterization and structural basis for the brightness of mCLIFY: a novel monomeric and circularly permuted bright yellow fluorescent protein.

We present mCLIFY: a monomeric, bright, yellow, and long-lived fluorescent protein (FP) created by circular permutation of YPet, the brightest yellow FP from Aequorea Victoria for use in cellular and in vitro single molecule studies. mCLIFY retains the enhanced photophysical properties of YPET as a monomer at concentrations ≤ 40 μM. In contrast, we determined that YPet has a dimerization dissociation constant (K D 1-2) of 3.4 μM. Dimerization of YPet can cause homo-FRET, which underlies quantitative errors due to dimerization and homo-FRET. We determined the atomic structure of mCLIFY at 1.57 Å resolution and used its similarity with Venus for guided chromophore-targeted substitution studies to provide insights into its enhanced photophysical properties. The mutation V58L within the chromophore pocket improved quantum yield and extinction coefficient, making mCLIFY ~30% brighter than Venus. The extensive characterization of the photophysical and structural properties of YPet and mCLIFY presented here allowed us to reveal the basis of their long lifetimes and enhanced brightness and the basis of YPet's dimerization.

Detrimental Increase of Spin-Phonon Coupling in Molecular Qubits on Substrates.

Molecular qubits are a promising platform for quantum information systems. Although single molecule and ensemble studies have assessed the performance of S = 1/2 molecules, it is understood that to function in devices, regular arrays of addressable qubits supported by a substrate are needed. The substrate imposes mechanical and electronic boundary conditions on the molecule; however, the impact of these effects on spin-lattice relaxation times is not well understood. Here we perform electronic structure calculations to assess the effects of a graphene (Cgr) substrate on the molecular qubit copper phthalocyanine (CuPc). We use a progressive Hessian approach to efficiently calculate and separate the substrate contributions. We also use a simple thermal model to predict the impact of these changes on the spin-phonon coupling from 0 to 200 K. Further analysis of the individual vibrational modes with and without Cgr shows that an overall increase in SPC between the vibrations modes of CuPc with the surface reduces the spin-lattice relaxation time T1. We explain these changes by examining how the substrate lifts symmetries of CuPc in the absorbed configuration. Our work shows that a surface can have a large unintentional impact on SPC and that ways to reduce this coupling need to be found to fully exploit arrays of molecular qubits in device architectures.

Positive feedback in Ras activation by full-length SOS arises from autoinhibition release mechanism

Signaling through the Ras-MAPK pathway can exhibit switch-like activation, which has been attributed to the underlying positive feedback and bimodality in the activation of RasGDP to RasGTP by SOS. SOS contains both catalytic and allosteric Ras binding sites, and a common assumption is that allosteric activation selectively by RasGTP provides the mechanism of positive feedback. However, recent single-molecule studies have revealed that SOS catalytic rates are independent of the nucleotide state of Ras in the allosteric binding site, raising doubt about this as a positive feedback mechanism. Here, we perform detailed kinetic analyses of receptor-mediated recruitment of full-length SOS to the membrane while simultaneously monitoring its catalytic activation of Ras. These results, along with kinetic modeling, expose the autoinhibition release step in SOS, rather than either recruitment or allosteric activation, as the underlying mechanism giving rise to positive feedback in Ras activation.

DNA-PK: A synopsis beyond synapsis

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not.Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK’s function in repair.

An atomic force microscopy and total internal reflection fluorescence microscopy correlated system (AFM-TIRF) for fluorescence imaging and spectroscopy of a single particle.

Combining atomic force microscopy (AFM) with other optical microscopic techniques is pivotal in nanoscale investigations, particularly leveraging the surface-sensitive properties of total internal reflection fluorescence microscopy (TIRF). A novel design that integrates AFM with a multi-wavelength TIRF is displayed, providing simultaneous fluorescence imaging and spectral acquisition capabilities. We elaborate on the considerations in the instrument design process and demonstrate the performance and potential applications of the instrument through fluorescence imaging and spectroscopy testing of individual nanoparticles. This AFM and TIRF correlated system (AFM-TIRF) emerges as a promising option for single-molecule fluorescence studies, enabling simultaneous manipulation and detection of fluorescence from individual molecules.

Making the Most of Nothing: One-Class Classification for Single-Molecule Transport Studies.

Single-molecule experiments offer a unique means to probe molecular properties of individual molecules-yet they rest upon the successful control of background noise and irrelevant signals. In single-molecule transport studies, large amounts of data that probe a wide range of physical and chemical behaviors are often generated. However, due to the stochasticity of these experiments, a substantial fraction of the data may consist of blank traces where no molecular signal is evident. One-class (OC) classification is a machine learning technique to identify a specific class in a data set that potentially consists of a wide variety of classes. Here, we examine the utility of two different types of OC classification models on four diverse data sets from three different laboratories. Two of these data sets were measured at cryogenic temperatures and two at room temperature. By training the models solely on traces from a blank experiment, we demonstrate the efficacy of OC classification as a powerful and reliable method for filtering out blank traces from a molecular experiment in all four data sets. On a labeled 4,4'-bipyridine data set measured at 4.2 K, we achieve an accuracy of 96.9 ± 0.3 and an area under the receiver operating characteristic curve of 99.5 ± 0.3 as validated over a fivefold cross-validation. Given the wide range of physical and chemical properties that can be probed in single-molecule experiments, the successful application of OC classification to filter out blank traces is a major step forward in our ability to understand and manipulate molecular properties.

Unraveling Burst Selection Bias in Single-Molecule FRET of Species with Unequal Brightness and Diffusivity.

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.

The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins -1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein-complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.

Effect of Environmental pH on the Mechanics of Chitin and Chitosan: A Single-Molecule Study.

Chitin and chitosan are important structural macromolecules for most fungi and marine crustaceans. The functions and application areas of the two molecules are also adjacent beyond their similar molecular structure, such as tissue engineering and food safety where solution systems are involved. However, the elasticities of chitin and chitosan in solution lack comparison at the molecular level. In this study, the single-molecule elasticities of chitin and chitosan in different solutions are investigated via atomic force microscope (AFM) based single-molecule spectroscopy (SMFS). The results manifest that the two macromolecules share the similar inherent elasticity in DOSM due to their same chain backbone. However, obvious elastic deviations can be observed in aqueous conditions. Especially, a lower pH value (acid environment) is helpful to increase the elasticity of both chitin and chitosan. On the contrary, the tendency of elastic variation of chitin and chitosan in a larger pH value (alkaline environment) shows obvious diversity, which is mainly determined by the side groups. This basic study may produce enlightenment for the design of intelligent chitin and chitosan food packaging and biomedical materials.

Single molecule studies of dynamic platelet interactions with endothelial cells.

A biotechnological platform consisting of two-color 3D super-resolution readout and a microfluidic system was developed to investigate platelet interaction with a layer of perfused endothelial cells under flow conditions. Platelet activation has been confirmed via CD62P clustering on the membrane and mitochondrial morphology of ECs at the single cell level were examined using 3D two-color single-molecule localization microscopy and classified applying machine learning. To compare binding of activated platelets to intact or stressed ECs, a femtosecond laser was used to induced damage to single ECs within the perfused endothelial layer. We observed that activated platelets bound to the perfused ECs layer preferentially in the proximity to single stressed ECs. Platelets activated under flow were ∼6 times larger compared to activated ones under static conditions. The CD62P expression indicated more CD62P proteins on membrane of dynamically activated platelets, with a tendency to higher densities at the platelet/EC interface. Platelets activated under static conditions showed a less pronounced CD62P top/bottom asymmetry. The clustering of CD62P in the platelet membrane differs depending on the activation conditions. Our results confirm that nanoscopic analysis using two-color 3D super-resolution technology can be used to assess platelet interaction with a stressed endothelium under dynamic conditions.

Compatibility of termination mechanisms in bacterial transcription with inference on eukaryotic models.

Transcription termination has evolved to proceed through diverse mechanisms. For several classes of terminators, multiple models have been debatably proposed. Recent single-molecule studies on bacterial terminators have resolved several long-standing controversies. First, termination mode or outcome is twofold rather than single. RNA is released alone before DNA or together with DNA from RNA polymerase (RNAP), i.e. with RNA release for termination, RNAP retains on or dissociates off DNA, respectively. The concomitant release, described in textbooks, results in one-step decomposition of transcription complexes, and this 'decomposing termination' prevails at ρ factor-dependent terminators. Contrastingly, the sequential release was recently discovered abundantly from RNA hairpin-dependent intrinsic terminations. RNA-only release allows RNAP to diffuse on DNA in both directions and recycle for reinitiation. This 'recycling termination' enables one-dimensional reinitiation, which would be more expeditious than three-dimensional reinitiation by RNAP dissociated at decomposing termination. Second, while both recycling and decomposing terminations occur at a hairpin-dependent terminator, four termination mechanisms compatibly operate at a ρ-dependent terminator with ρ in alternative modes and even intrinsically without ρ. RNA-bound catch-up ρ mediates recycling termination first and decomposing termination later, while RNAP-prebound stand-by ρ invokes only decomposing termination slowly. Without ρ, decomposing termination occurs slightly and sluggishly. These four mechanisms operate on distinct timescales, providing orderly fail-safes. The stand-by mechanism is benefited by terminational pause prolongation and modulated by accompanying riboswitches more greatly than the catch-up mechanisms. Conclusively, any mechanism alone is insufficient to perfect termination, and multiple mechanisms operate compatibly to achieve maximum possible efficiency under separate controls.

Eukaryotic yeast V1-ATPase rotary mechanism insights revealed by high-resolution single-molecule studies.

Vacuolar ATP-dependent proton pumps (V-ATPases) belong to a super-family of rotary ATPases and ATP synthases. The V1 complex consumes ATP to drive rotation of a central rotor that pumps protons across membranes via the Vo complex. Eukaryotic V-ATPases are regulated by reversible disassembly of subunit C, V1 without C, and VO. ATP hydrolysis is thought to generate an unknown rotary state that initiates regulated disassembly. Dissociated V1 is inhibited by subunit H that traps it in a specific rotational position. Here, we report the first single-molecule studies with high resolution of time and rotational position of Saccharomyces cerevisiae V1-ATPase lacking subunits H and C (V1ΔHC), which resolves previously elusive dwells and angular velocity changes. Rotation occurred in 120° power strokes separated by dwells comparable to catalytic dwells observed in other rotary ATPases. However, unique V1ΔHC rotational features included: 1) faltering power stroke rotation during the first 60°; 2) a dwell often occurring ∼45° after the catalytic dwell, which did not increase in duration at limiting MgATP; 3) a second dwell, ∼2-fold longer occurring 112° that increased in duration and occurrence at limiting MgATP; 4) limiting MgATP-dependent decreases in power stroke angular velocity where dwells were not observed. The results presented here are consistent with MgATP binding to the empty catalytic site at 112° and MgADP released at ∼45°, and provide important new insight concerning the molecular basis for the differences in rotary positions of substrate binding and product release between V-type and F-type ATPases.

A Biophysics Toolbox for Reliable Data Acquisition and Processing in Integrated Force-Confocal Fluorescence Microscopy.

Integrated single-molecule force-fluorescence spectroscopy setups allow for simultaneous fluorescence imaging and mechanical force manipulation and measurements on individual molecules, providing comprehensive dynamic and spatiotemporal information. Dual-beam optical tweezers (OT) combined with a confocal scanning microscope form a force-fluorescence spectroscopy apparatus broadly used to investigate various biological processes, in particular, protein:DNA interactions. Such experiments typically involve imaging of fluorescently labeled proteins bound to DNA and force spectroscopy measurements of trapped individual DNA molecules. Here, we present a versatile state-of-the-art toolbox including the preparation of protein:DNA complex samples, design of a microfluidic flow cell incorporated with OT, automation of OT-confocal scanning measurements, and the development and implementation of a streamlined data analysis package for force and fluorescence spectroscopy data processing. Its components can be adapted to any commercialized or home-built dual-beam OT setup equipped with a confocal scanning microscope, which will facilitate single-molecule force-fluorescence spectroscopy studies on a large variety of biological systems.

Mechanistic Insight into the Mechanical Unfolding of the Integral Membrane Diacylglycerol Kinase.

The essential forces stabilizing membrane proteins and governing their folding and unfolding are difficult to decipher. Single-molecule atomic force spectroscopy mechanically unfolds individual membrane proteins and quantifies their dynamics and energetics. However, it remains challenging to structurally assign unfolding intermediates precisely and to deduce dominant interactions between specific residues that facilitate either the localized stabilization of these intermediates or the global assembly of membrane proteins. Here, we performed force spectroscopy experiments and multiscale molecular dynamics simulations to study the unfolding pathway of diacylglycerol kinase (DGK), a small trimeric multispan transmembrane enzyme. The remarkable agreement between experiments and simulations allowed precise structural assignment and interaction analysis of unfolding intermediates, bypassing existing limitations on structural mapping, and thus provided mechanistic explanations for the formation of these states. DGK unfolding was found to proceed with structural segments varying in size that do not correlate with its secondary structure. We identified intermolecular side-chain packing interactions as one of the major contributions to the stability of unfolding intermediates. Mutagenesis creating packing defects induced a dramatic decrease in the mechano-stability of corresponding intermediates and also in the thermo-stability of DGK trimer, in good agreement with predictions from simulations. Hence, the molecular determinants of the mechano- and thermo-stability of a membrane protein can be identified at residue resolution. The accurate structural assignment established and microscopic mechanism revealed in this work may substantially expand the scope of single-molecule studies of membrane proteins.

Supra-biological performance of immobilized enzymes enabled by chaperone-like specific non-covalent interactions

Designing complex synthetic materials for enzyme immobilization could unlock the utility of biocatalysis in extreme environments. Inspired by biology, we investigate the use of random copolymer brushes as dynamic immobilization supports that enable supra-biological catalytic performance of immobilized enzymes. This is demonstrated by immobilizing Bacillus subtilis Lipase A on brushes doped with aromatic moieties, which can interact with the lipase through multiple non-covalent interactions. Incorporation of aromatic groups leads to a 50 °C increase in the optimal temperature of lipase, as well as a 50-fold enhancement in enzyme activity. Single-molecule FRET studies reveal that these supports act as biomimetic chaperones by promoting enzyme refolding and stabilizing the enzyme’s folded and catalytically active state. This effect is diminished when aromatic residues are mutated out, suggesting the importance of π-stacking and π-cation interactions for stabilization. Our results underscore how unexplored enzyme-support interactions may enable uncharted opportunities for using enzymes in industrial biotransformations.