Single-molecule Fluorescence Tracking Research Articles

THU572 Structure, Organisation And Mechanistic Insight Of Cytochrome P450 17A1

Abstract Disclosure: L.L. Martin: None. P. O'Leary: None. S. Arumugam: None. S. Bell: None. R.J. Rodgers: None. Introduction: Cytochrome P45017A1 (CYP17) is a heme-containing, membrane bound enzyme situated at a key branch within steroidogenic pathways. CYP17 is a dual function enzyme that initially catalyses 17α-hydroxylation of pregnenolone and progesterone to 17α-hydroxypregnenolone and 17α-hydroxyprogesterone. CYP17 can further metabolize these steroids via 17,20-lyase reactions to produce androgen/estrogen precursors. Activity of CYP17 requires electrons delivered by cytochrome P450 oxido-reductase (CPR) and and accessory protein cytochrome b5 (cyt b5) for the 17,20-lyase reaction. Previously, we showed that (i) CYP17 could homodimerise and heterodimerise with CPR and cyt b5 in cells using FRET [1] and computer models [2] and (ii) protein-protein interaction of cyt b5 with CYP17 slowed the rate of electron transfer to CYP17 [3] by electrochemistry. The current study focuses on sub-cellular organisation of CYP17 and mechanistic insight gained via analysis of the structural and molecular interactions of CYP17 with its partner proteins associated with 17α-hydroxylase and 17,20-lyase reactions. Methods: Single molecule fluorescence microscopy (SMFM) was used to observe fluorescently tagged CYP17 in live HEK 293 cells. Molecular Dynamics (MD) simulations were done for CYP17 in membranes, in the presence and absence of substrates, while interacting with CPR and cyt b5. Results: Halo-tagged CYP17 was imaged in real-time within the endoplasmic reticulum, providing kinetic data and sub-cellular localisation dynamics of individual molecules.MD simulations, within a membrane environment, allowed the dynamics of full-length models of CYP17 to be simulated: (i) in the absence of substrate, (ii) with a hydroxylase substrate (pregnenolone), and (iii) with a lyase substrate (17α-OH pregnenolone). Intriguingly, the protein-protein interaction of cyt b5 decreased the movement and flexibility within the active site of CYP17, however, regions not necessarily obvious for haem-substrate rearrangement were also altered. Single molecule fluorescence revealed CYP17 molecules moved dynamically along the endoplasmic reticulum. Conclusions: MD analyses provided structural insight on the mechanism underlying the allosteric coupling between the cyt b5 to the CYP17 interface and specifically to the active site. Single molecule fluorescence tracking indicated that CYP17 actively moves dynamically in the endoplasmic reticulum. 1. Praporski, S., et al., Organization of Cyt P450 Enzymes Involved in Sex Steroid Synthesis. J. Biol. Chem., 2009. 284 33224-33232. 2. Holien, J.K., et al., A Homodimer Model Can Resolve the Conundrum as to How CPR and Cyt b5 Compete for the Same Binding Site on Cyt P450c17. Curr Protein Pept Sci, 2017. 18 515-521. 3. Simonov, A.N., et al., Mechanistic Scrutiny Identifies a Kinetic Role for Cyte b5 Regulation of Human Cyt P450c17. PLoS ONE, 2015. 10 e0141252. Presentation Date: Thursday, June 15, 2023

Interfacial diffusion of single polyethylene oxide molecules: the consistency between fluorescence correlation spectroscopy and single molecule fluorescence tracking

高分子表界面的扩散运动在决定体系流变学性质、界面化学反应速率、相分离及其动力学、生物功能与信号传导等发挥着关键作用，因而，有效、准确测量表界面处单个高分子的扩散运动具有十分重要的意义. 本文以荧光关联光谱、单分子荧光轨迹追踪技术为手段，以吸附于固液界面的聚乙二醇（PEO）分子为模型体系，对上述两种研究方法进行了详细的比较. 结果表明，虽然两种方法原理不同，覆盖的时间窗口不同，测量空间范围不同，但是在合适的测量时间窗口内，对理想界面（水/硅烷化单晶薄膜的界面）处的PEO单分子扩散运动测量，获得了一致的扩散系数结果，表明了两种研究方法对于理想界面处单分子扩散测量的准确性与可靠性.

Resolving the kinetics of transcription factor recognition of the target DNA site via advanced single-molecule FRET spectroscopy.

Transcription factors function by specifically binding to target DNA sites to initiate gene expression. Such DNA recognition process usually involves an interplay between high-affinity binding to the cognate sequence and low-affinity binding to any other DNA. The latter enables scanning of the DNA via a 1D diffusive process, which combined with 3D diffusion-collision kinetics, facilitates target location. 1D diffusion has been observed using single-molecule fluorescence tracking on many DNA binding proteins (DPBs). Powerful experimental methods are also available to determine the sequence logo recognized by any given DBP. However, there are no approaches available that resolve the kinetics of locking into the target site, which is essential to elucidate the mechanisms of cognate DNA recognition. Here we introduce such an approach, by using single-molecule FRET spectroscopy and maximum likelihood analysis of photon arrival times. We apply it to determine the rates of lock-into and release-from the target site of the Engrailed homeodomain (EnHD). To resolve between non-specific and cognate DNA binding we exploit the recent discovery that EnHD changes conformation upon cognate binding, resulting in a large shift in transfer efficiency between a donor-acceptor pair placed at the protein ends. Our experiments on a 21-bp dsDNA containing the EnHD cognate sequence reveal that EnHD finds its target in few milliseconds. Remarkably, the on-rate at the C50 is mostly invariant over orders of magnitude differences in ionic strength and DNA concentration. This is because the changes in affinity caused by ionic strength are mostly recapitulated on the off-rate, which is the exact opposite to diffusion-control kinetics. We hence conclude that EnHD's lock-into target kinetics are controlled by its conformational dynamics, which makes the response time for controlling transcription only dependent on site occupancy levels.

Diffusion of Short Semiflexible DNA Polymer Chains in Strong and Moderate Confinement.

In many technological applications, DNA is confined within nanoenvironments that are smaller than the size of the unconfined polymer in solution. However, the dependence of the diffusion coefficient on molecular weight and characteristic confinement dimension remains poorly understood in this regime. Here, convex lens-induced confinement (CLiC) was leveraged to examine how the diffusion of short DNA fragments varied as a function of slit height by using single-molecule fluorescence tracking microscopy. The diffusion coefficient followed approximate power law behavior versus confinement height, with exponents of 0.27 ± 0.01, 0.32 ± 0.02, and 0.42 ± 0.06 for 692, 1343, and 2686 base pair chains, respectively. The weak dependence on slit height suggests that shorter semiflexible chains may adopt increasingly rodlike conformations and therefore experience weaker excluded-volume interactions as the confinement dimension is reduced. The diffusion coefficient versus molecular weight also exhibited apparent power law behavior, with exponents that varied slightly (from -0.89 to -0.85) with slit height, consistent with hydrodynamic interactions intermediate between Rouse and Zimm model predictions.

Faster Surface Ligation Reactions Improve Immobilized Enzyme Structure and Activity.

During integration into materials, the inactivation of enzymes as a result of their interaction with nanometer size denaturing "hotspots" on surfaces represents a critical challenge. This challenge, which has received far less attention than improving the long-term stability of enzymes, may be overcome by limiting the exploration of surfaces by enzymes. One way this may be accomplished is through increasing the rate constant of the surface ligation reaction and thus the probability of immobilization with reactive surface sites (i.e., ligation efficiency). Here, the connection between ligation reaction efficiency and the retention of enzyme structure and activity was investigated by leveraging the extremely fast reaction of strained trans-cyclooctene (sTCOs) and tetrazines (Tet). Remarkably, upon immobilization via Tet-sTCO chemistry, carbonic anhydrase (CA) retained 77% of its solution-phase activity, while immobilization via less efficient reaction chemistries, such as thiol-maleimide and azide-dibenzocyclooctyne, led to activity retention of only 46% and 27%, respectively. Dynamic single-molecule fluorescence tracking methods further revealed that longer surface search distances prior to immobilization (>0.5 μm) dramatically increased the probability of CA unfolding. Notably, the CA distance to immobilization was significantly reduced through the use of Tet-sTCO chemistry, which correlated with the increased retention of structure and activity of immobilized CA compared to the use of slower ligation chemistries. These findings provide an unprecedented insight into the role of ligation reaction efficiency in mediating the exploration of denaturing hotspots on surfaces by enzymes, which, in turn, may have major ramifications in the creation of functional biohybrid materials.

Quantitative spectroscopy of single molecule interaction times.

Single molecule fluorescence tracking provides information at nanometer-scale and millisecond-temporal resolution about the dynamics and interaction of individual molecules in a biological environment. While the dynamic behavior of isolated molecules can be characterized well, the quantitative insight is more limited when interactions between two indistinguishable molecules occur. We address this aspect by developing a theoretical foundation for a spectroscopy of interaction times, i.e., the inference of interaction from imaging data. A non-trivial crossover between a power law to an exponential behavior of the distribution of the interaction times is highlighted, together with the dependence of the exponential term upon the microscopic reaction affinity. Our approach is validated with simulated and experimental datasets.

The disordered DNA-binding domain of p53 is indispensable for forming an encounter complex to and jumping along DNA

The tumor suppressor p53 utilizes a facilitated diffusion mechanism to search for and bind to target DNA sequences. Sub-millisecond single-molecule fluorescence tracking demonstrated that p53 forms a short-lived encounter complex to DNA then converts to the long-lived complex that can move and jump along DNA during the target search. To reveal the role of each DNA-binding domain of p53 in these processes, we investigated two p53 mutants lacking either of two DNA-binding domains; structured core and disordered C-terminal domains, using sub-millisecond single-molecule fluorescence microscopy. We found that the C-terminal domain is required for the encounter complex formation and conversion to the long-lived complex. The long-lived complex is stabilized by the core domain as well as the C-terminal domain. Furthermore, only the C-terminal domain participates in the jump of p53 along DNA at a high salt concentration. We propose that the flexible C-terminal domain of p53 is twined around DNA, which can form the encounter complex, convert to the long-lived complex, and enable p53 to land on DNA after the jump.

Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking

Characterization of the target search dynamics of DNA-binding proteins along DNA has been hampered by the time resolution of a standard single-molecule fluorescence microscopy. Here, we achieved the time resolution of 0.5 ms in the fluorescence microscopy measurements by optimizing the fluorescence excitation based on critical angle illumination and by utilizing the time delay integration mode of the electron-multiplying charge coupled device. We characterized the target search dynamics of the tumor suppressor p53 along nonspecific DNA at physiological salt concentrations. We identified a short-lived encounter intermediate before the formation of the long-lived p53–DNA complex. Both the jumps and the one-dimensional diffusion of p53 along DNA were accelerated at higher salt concentrations, suggesting the rotation-uncoupled movement of p53 along DNA grooves and conformational changes in the p53/DNA complex. This method can be used to clarify the unresolved dynamics of DNA-binding proteins previously hidden by time averaging.

Transient Binding and Non-rotational Coupled Motion of p53 Revealed by Sub-Millisecond Resolved Single-Molecule Fluorescence Tracking

Target specific DNA-binding proteins (DBP) are required to search for and bind to their target DNA sequence quickly and accurately using various strategies such as 1D diffusion and 3D jump. However, many more events have been postulated in the target search dynamics. For example, an encounter complex between DBP and DNA, assumed to be present before the formation of the stable DNA-DBP complex, is proposed to allow DBP to be in the proximity of DNA and promote the target search. However, the limited time resolution of the previous methods, several tens of milliseconds, hampers the distinction of these motions.

CpG and methylation-dependent DNA binding and dynamics of the methylcytosine binding domain 2 protein at the single-molecule level.

The methylcytosine-binding domain 2 (MBD2) protein recruits the nucleosome remodeling and deacetylase complex (NuRD) to methylated DNA to modify chromatin and regulate transcription. Importantly, MBD2 functions within CpG islands that contain 100s to 1000s of potential binding sites. Since NuRD physically rearranges nucleosomes, the dynamic mobility of this complex is directly related to function. In these studies, we use NMR and single-molecule atomic force microscopy and fluorescence imaging to study DNA binding dynamics of MBD2. Single-molecule fluorescence tracking on DNA tightropes containing regions with CpG-rich and CpG-free regions reveals that MBD2 carries out unbiased 1D diffusion on CpG-rich DNA but subdiffusion on CpG-free DNA. In contrast, the protein stably and statically binds to methylated CpG (mCpG) regions. The intrinsically disordered region (IDR) on MBD2 both reduces exchange between mCpG sites along the DNA as well as the dissociation from DNA, acting like an anchor that restricts the dynamic mobility of the MBD domain. Unexpectedly, MBD2 binding to methylated CpGs induces DNA bending that is augmented by the IDR region of the protein. These results suggest that MBD2 targets NuRD to unmethylated or methylated CpG islands where its distinct dynamic binding modes help maintain open or closed chromatin, respectively.

Walking the DNA Methylation Tightrope: The Involvement of Intrinsically Disordered Regions of Transcription Factors

Most 5-methylcytosine-binding domain proteins (such as MeCP2 and MBD1-6) contain a highly conserved 70 amino acid methyl-CpG-binding domain (MBD). The founding member of vertebrate MBD proteins, MBD2, is associated with the Nucleosome Remodeling Deacetylase complex (NuRD) and exhibits DNA binding specificity for methylated CpG (mCpG) islands. MBD2 consists of the MBD, an intrinsically disorder region (IDR), and a coiled-coil region. The inclusion of the IDR in MBD2 increases binding affinity for mCpG. However, how MBD2 recognizes methylated DNA and the role of the IDR in influencing binding are not fully understood. To further elucidate the role of the IDR in DNA binding, we incorporated a series of biophysical techniques to examine movement of MBD2 without the IDR (MBD2MBD) or with a portion of the IDR (MBD2MBD+IDR). NMR and ZZ-exchange spectroscopy showed differences in the distribution and intramolecular exchange rate of the two protein constructs on 17bp dsDNA substrates that contain either CpG islands, mCpG sites, or no CpG sites. Single-molecule fluorescence tracking of quantum dot-labeled MBD2MBD and MBD2MBD+IDR on DNA tightropes containing CpG or mCpG reveals that both MBD2MBD and MBD2MBD+IDR search on DNA through 1D free diffusion and stably bind to methylated DNA. In conclusion, our data reveals how MBD2 dynamically achieves DNA binding specificity for methylated DNA and supports that the IDR of MBD2 modulates MBD2-binding dynamics. These results are important for further deciphering the molecular mechanisms of MBD and mCpG epigenetic regulation.

Membrane Crowding and Anomalous Diffusion in Artificial Lipid Bilayers

In contrast to membrane protein diffusion in artificial bilayers, the diffusion of proteins in cells is both slower and non-ergodic. Anomalous diffusion in cells has been attributed to cytoskeletal-membrane interactions and nanoscopic heterogeneity in membrane protein and lipid composition. Using a combination of single-molecule fluorescence tracking and interferometric scattering microscopy we quantify how these factors affect anomalous diffusion over a range of time-scales in artificial lipid membranes. By varying the membrane composition and nature and density of pinning sites, we examine the onset of anomalous diffusion and its dependence on these properties. We observe a reduction in tracer diffusion rates as membrane crowding is increased, and the diffusion appears to become increasingly anomalous as membrane components are immobilized.

Dynamics of Myosin XI: The Family Speed Demon

Myosin XI is the fastest known processive motor, and is implicated in organelle transport and propulsion of cytoplasmic streaming in plant cells. We studied the motility and dynamics of a recombinant myosin XI motor domain hybridized with the myosin Va lever arm with 6 calmodulin-bearing IQ motifs and the dimerization motif. Single molecule fluorescence tracking (FIONA) and polarization (polTIRF) measurements support the hand-over-hand stepping model for myosin XI. Its step size (∼34.5 nm) is slightly shorter than the helical pitch of actin filaments, suggesting an overall left-handed helical walking path. The path was confirmed by polTIRF azimuthal angles as well as bead motility on suspended actin filaments. The hybrid myosin XI showed a maximal velocity of at least 4 μm/s, which is ∼7-fold faster than its structurally similar cousin, myosin V, but they have similar run lengths of ∼1 μm. FIONA measurements showed faster myosin XI stepping rates on actin filaments as compared to bundled actin. The gliding filament assay gave 5-10-fold slower velocities than single molecule processive runs. PolTIRF experiments identified several classes of molecules, with different leading and trailing probe angles. These classes may represent placement of the bifunctional rhodmaine probe on different calmodulins. Compared with myosin V, myosin XI showed more variable localization between steps and more off-axis motion, which may relate to myosin XI's role in cytoplasmic streaming on bundled actin beams. Supported by NIH grant GM086352.

Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging

The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field.

Tracking of Single Receptor Molecule Mobility in Neuronal Membranes: A Quick Theoretical and Practical Guide

Single-molecule detection enables us to visualise the real-time dynamics of individual molecules in live cells. We review the recent advancements in single-molecule fluorescence tracking of receptor protein mobility in the neuronal membrane. First, we discuss the practical consideration of single-molecule tracking in neurones, including the choice of cells and possible fluorescent labelling, as well as the appropriate optical set-up and imaging technology. We then describe the analysis of the single-molecule imaging data, including its theoretical and practical aspects of and relevant estimations of the biophysical parameters. Finally, we provide an example of a single-molecule tracking study in neuroendocrinology and highlight the next frontiers of single-molecule detection technologies.

Accelerating the Phase Separation in Aqueous Poly(N-isopropylacrylamide) Solutions by Slight Modification of the Polymer Stereoregularity: A Single Molecule Fluorescence Study

We discovered for aqueous thermoresponsive polymer solutions that only a slight change in stereoregularity of the polymer can drastically accelerate phase separation. Single molecule fluorescence tracking (SMT) for an isotactic-slight-rich (meso-diad-rich) polymer sample solution revealed an interpolymer nanonetwork even before phase separation, and also revealed a novel phase in which translational molecular motion was frozen after phase separation. For such systems, fluorescence correlation spectroscopy (FCS) provided quantitative information on molecular diffusion. The results on FCS well agreed with the interpolymer nanonetwork model that was proposed on the basis of SMT measurement. We demonstrate such a novel method to control phase separation dynamics and also the interpolymer nanonetwork model.

A novel method for automatic single molecule tracking of blinking molecules at low intensities

Single molecule tracking provides unprecedented insights into diffusional processes of systems in life and material sciences. Determination of molecule positions with high accuracy and correct connection of the determined positions to tracks is a challenging task with, so far, no universal solution for single fluorescing molecules tackling the challenge of low signal-to-noise ratios, frequent blinking and photo bleaching. Thus, the development of novel algorithms for automatic single molecule fluorescence tracking is essential to analyse the huge amount of diffusional data obtained with single molecule widefield fluorescence microscopy. Here, we present a novel tracking model using a top-down polyhedral approach which can be implemented effectively using standard linear programming solvers. The results of our tracking approach are compared to the ground truth of simulated data with different diffusion coefficients, signal-to-noise ratios and particle densities. We also determine the dependency of blinking on the analysed distribution of diffusion coefficients. To confirm the functionality of our tracking method, the results of automatic tracking and manual tracking by a human expert are compared and discussed.

A study on the mechanism of RecA in homologous recognition by using single molecule fluorescence tracking

RecA plays an important role in homologous recognition in prokaryotes, and it has become a hot point in homologous recognition related research since its discovery. We establish an assay by combining total internal reflection fluorescence and flow stretching to visualize in real time the motion of single RecA-ssDNA filaments which are tagged with fluorescent beads. This enables us to study the interaction of RecA-ssDNA filaments with their templates in the homologous recognition process. It is found that the searching and binding is a short-time (τ=0.2 s) and short-distance (l=1.05 μm) process. Two distinguished motion modes for the RecA-ssDNA filament are observed, a Brownian motion and a directed motion. The observations suggest a model that a RecA-ssDNA filament just interacts weakly with the template DNA before it binds firmly to the template DNA. If no homologous site is found in a searching process, the filament drops off the template and repeats the searching process again until it finally finds its target.

Bayesian Inference for Improved Single Molecule Fluorescence Tracking

Single molecule tracking is widely used to monitor the change in position of lipids and proteins in living cells. In many experiments in which molecules are tagged with a single or small number of fluorophores, the signal/noise ratio may be limiting, the number of molecules is not known, and fluorophore blinking and photobleaching can occur. All these factors make accurate tracking over long trajectories difficult and hence there is still a pressing need to develop better algorithms to extract the maximum information from a sequence of fluorescence images. We describe here a Bayesian-based inference approach, based on a trans-dimensional sequential Monte Carlo method that utilizes both the spatial and temporal information present in the image sequences. We show, using model data, where the real trajectory of the molecule is known, that our method allows accurate tracking of molecules over long trajectories even with low signal/noise ratio and in the presence of fluorescence blinking and photobleaching. The method is then applied to real experimental data.

Nanopipette Delivery of Individual Molecules to Cellular Compartments for Single-Molecule Fluorescence Tracking

We have developed a new method, using a nanopipette, for controlled voltage-driven delivery of individual fluorescently labeled probe molecules to the plasma membrane which we used for single-molecule fluorescence tracking (SMT). The advantages of the method are 1), application of the probe to predefined regions on the membrane; 2), release of only one or a few molecules onto the cell surface; 3), when combined with total internal reflection fluorescence microscopy, very low background due to unbound molecules; and 4), the ability to first optimize the experiment and then repeat it on the same cell. We validated the method by performing an SMT study of the diffusion of individual membrane glycoproteins labeled with Atto 647-wheat germ agglutin in different surface domains of boar spermatozoa. We found little deviation from Brownian diffusion with a mean diffusion coefficient of 0.79 ± 0.04 μm 2/s in the acrosomal region and 0.10 ± 0.02 μm 2/s in the postacrosomal region; this difference probably reflects different membrane structures. We also showed that we can analyze diffusional properties of different subregions of the cell membrane and probe for the presence of diffusion barriers. It should be straightforward to extend this new method to other probes and cells, and it can be used as a new tool to investigate the cell membrane.