Microscopes for fluorimeters: the era of single molecule measurements.
Ontology highlight
ABSTRACT:
SUBMITTER: Vale RD
PROVIDER: S-EPMC2762818 | biostudies-literature | 2008 Nov
REPOSITORIES: biostudies-literature
Publications
Microscopes for fluorimeters: the era of single molecule measurements.
Cell 20081101 5
As transforming as the first atomic resolution view of myoglobin in the late 1950s, scientists can now use a suite of single molecule technologies to watch protein macromolecular machines executing their functions "in real time." This Essay highlights applications and challenges of single molecule studies in structural biology, cell biology, and biotechnology. ...[more]
Similar Datasets
Project description:Genome packaging of double-stranded RNA (dsRNA) phages has been widely studied using biochemical and molecular biology methods. We adapted the existing in vitro packaging system of one such phage for single-molecule experimentation. To our knowledge, this is the first attempt to study the details of viral RNA packaging using optical tweezers. Pseudomonas phage φ6 is a dsRNA virus with a tripartite genome. Positive-sense (+) single-stranded RNA (ssRNA) genome precursors are packaged into a preformed procapsid (PC), where negative strands are synthesized. We present single-molecule measurements of the viral ssRNA packaging by the φ6 PC. Our data show that packaging proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Although the mean packaging velocity was relatively low (0.07-0.54 nm/sec), packaging could reach 4.62 nm/sec during the fast packaging phase.
| S-EPMC5159644 | biostudies-literature
Project description:Single-molecule methods provide direct measurements of macromolecular dynamics, but are limited by the number of degrees of freedom that can be followed at one time. High-resolution rotor bead tracking (RBT) measures DNA torque, twist, and extension, and can be used to characterize the structural dynamics of DNA and diverse nucleoprotein complexes. Here, we extend RBT to enable simultaneous monitoring of additional degrees of freedom. Fluorescence-RBT (FluoRBT) combines magnetic tweezers, infrared evanescent scattering, and single-molecule FRET imaging, providing real-time multiparameter measurements of complex molecular processes. We demonstrate the capabilities of FluoRBT by conducting simultaneous measurements of extension and FRET during opening and closing of a DNA hairpin under tension, and by observing simultaneous changes in FRET and torque during a transition between right-handed B-form and left-handed Z-form DNA under controlled supercoiling. We discover unanticipated continuous changes in FRET with applied torque, and also show how FluoRBT can facilitate high-resolution FRET measurements of molecular states, by using a mechanical signal as an independent temporal reference for aligning and averaging noisy fluorescence data. By combining mechanical measurements of global DNA deformations with FRET measurements of local conformational changes, FluoRBT will enable multidimensional investigations of systems ranging from DNA structures to large macromolecular machines.
| S-EPMC5984952 | biostudies-literature
Project description:We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G?GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA?EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.
| S-EPMC3090999 | biostudies-literature
Project description:We use single-molecule force clamp spectroscopy (SMFCS) to explore the reactivity of tris(2-carboxyethyl)phosphine (TCEP), 1, 4-dl-dithiothreitol (DTT) and hydrosulfide anion (HS(-)) on disulfide bonds within a mechanically stretched polypeptide. The single-bond level bimolecular nucleophilic substitution (S(N)2) events are recorded at a series of precisely controlled temperatures so that the Arrhenius kinetic parameters, that is, the height of the activation energy barrier (E(a)) and the attempting frequency (A) of the chemical reactions, can be determined. The values of A are typically at the order of 10(7) M(-1) s(-1), which is far lower than that predicted by the transition-state theory, in which A is given by k(B)T/h and around 10(12) M(-1) s(-1) at room temperature. Furthermore, E(a) is derived to be 30-40 kJ/mol, which can be lowered by ?6-8% with every 100 pN mechanical force applied. The correlation of the A and E(a) with the molecular structures reveals that the relative magnitude of these two parameters cannot be simply judged from the size of the molecule or the nucleophilicity of the attacking atom. The comparison of the influences on the reaction rate induced by force and temperature indicates an equivalent accelerating effect by every 50 pN or 10 K increment, giving for the first time the relationship between mechanical and thermal effects on a single-molecule S(N)2 chemical reaction.
| S-EPMC3711696 | biostudies-literature
Project description:Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.
| S-EPMC2885172 | biostudies-literature
Project description:DNA is an attractive material for a range of applications in nanoscience and nanotechnology, and it has recently been demonstrated that the electronic properties of DNA are uniquely sensitive to its sequence and structure, opening new opportunities for the development of electronic DNA biosensors. In this report, we examine the origin of multiple conductance peaks that can occur during single-molecule break-junction (SMBJ)-based conductance measurements on DNA. We demonstrate that these peaks originate from the presence of multiple DNA conformations within the solutions, in particular, double-stranded B-form DNA (dsDNA) and G-quadruplex structures. Using a combination of circular dichroism (CD) spectroscopy, computational approaches, sequence and environmental controls, and single-molecule conductance measurements, we disentangle the conductance information and demonstrate that specific conductance values come from specific conformations of the DNA and that the occurrence of these peaks can be controlled by controlling the local environment. In addition, we demonstrate that conductance measurements are uniquely sensitive to identifying these conformations in solutions and that multiple configurations can be detected in solutions over an extremely large concentration range, opening new possibilities for examining low-probability DNA conformations in solutions.
| S-EPMC9855376 | biostudies-literature
Project description:Magnetic tweezers (MT) are single-molecule manipulation instruments that utilize a magnetic field to apply force to a biomolecule-tethered magnetic bead while using optical bead tracking to measure the biomolecule's extension. While relatively simple to set up, prior MT implementations have lacked the resolution necessary to observe sub-nanometer biomolecular configuration changes. Here, we demonstrate a reflection-interference technique for bead tracking, and show that it has much better resolution than traditional diffraction-based systems. We enhance the resolution by fabricating optical coatings on all reflecting surfaces that optimize the intensity and contrast of the interference image, and we implement feedback control of the focal position to remove drift. To test the system, we measure the length change of a DNA hairpin as it undergoes a folding/unfolding transition.
| S-EPMC2777445 | biostudies-literature
Project description:Single molecule photobleaching is a powerful technique to measure the number of fluorescent units in subresolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photobleaching can occur before the sample is appropriately placed and focused. Such "prebleaching" can introduce a strong systematic bias toward smaller oligomers. Quantitative correction of prebleaching is known to be an ill-posed problem, limiting the utility of the technique. Here, we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the prebleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human islet amyloid polypeptide. Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.
| S-EPMC7063439 | biostudies-literature
Project description:An important measure of the conformation of protein molecules is the degree of surface exposure of its specific segments. However, this is hard to measure at the level of individual molecules. Here, we combine single molecule photobleaching (smPB, which resolves individual photobleaching steps of single molecules) and fluorescence quenching techniques to measure the accessibility of individual fluorescently labeled protein molecules to quencher molecules in solution. A quencher can reduce the time a fluorophore spends in the excited state, increasing its photostability under continuous irradiation. Consequently, the photo-bleaching step length would increase, providing a measure for the accessibility of the fluorophore to the solvent. We demonstrate the method by measuring the bleaching step-length increase in a lipid, and also in a lipid-anchored peptide (both labelled with rhodamine-B and attached to supported lipid bilayers). The fluorophores in both molecules are expected to be solvent-exposed. They show a near two-fold increase in the step length upon incubation with 5 mM tryptophan (a quencher of rhodamine-B), validating our approach. A population distribution plot of step lengths before and after addition of tryptophan show that the increase is not always homogenous. Indeed there are different species present with differential levels of exposure. We then apply this technique to determine the solvent exposure of membrane-attached N-terminus labelled amylin (h-IAPP, an amyloid associated with Type II diabetes) whose interaction with lipid bilayers is poorly understood. hIAPP shows a much smaller increase of the step length, signifying a lower level of solvent exposure of its N-terminus. Analysis of results from individual molecules and step length distribution reveal that there are at least two different conformers of amylin in the lipid bilayer. Our results show that our method ("Q-SLIP", Quenching-induced Step Length increase in Photobleaching) provides a simple route to probe the conformational states of membrane proteins at a single molecule level.
| S-EPMC8672140 | biostudies-literature
Project description:The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.
| S-EPMC4643199 | biostudies-literature