Single Molecule Spectroscopic Methods Research Articles

Investigating local DNA conformational trapping dynamics during DNA polymerase holoenzyme assembly and exonuclease proof-reading activity.

The sugar-phosphate backbones of DNA continuously undergo thermally-activated conformational fluctuations at equilibrium, which directly affect base-pair unstacking and hydrogen bond breaking. The influence of these motions on biomolecular function are particularly important at single-stranded (ss)—double-stranded (ds) DNA forks and primer-template (p/t) junctions. At these junctions, non-canonical structures that deviate from the Watson-Crick B-form DNA duplex may serve as recognition sites for protein binding, and the kinetics of these interactions likely play a central regulatory role for fundamental biological processes such as DNA replication and repair. The DNA polymerase/sliding clamp holoenzyme is the central protein complex that carries out the process of complementary nucleotide addition to nascent ssDNA template strands initiated at p/t junctions. In addition to playing a role in protein assembly, local DNA conformations resulting from nucleotide misincorporation regulate the exonuclease proof-reading activity of DNA polymerase. To study the details of these processive local DNA conformational dynamics, our group has developed a molecular probing method that relies on the rigid insertion of two fluorescent cyanine (Cy3) probes at opposite positions within the sugar-phosphate backbones of complementary DNA strands. Electrostatic coupling between probes results in delocalized optical properties that sensitively depend on relative probe orientations. Utilizing a series of complementary ensemble-based (i.e., fluorescence, absorbance, circular dichroism) and single-molecule spectroscopic methods, we study the local DNA backbone conformational dynamics as they relate to protein binding and activity. In this presentation, I will discuss recent studies of DNA polymerase holoenzyme assembly and exonuclease proofreading activity at p/t junctions. Specifically, results from microsecond-resolved polarization-sweep single-molecule spectroscopy (PS-SMS) demonstrate how protein-dependent conformational trapping of otherwise sparsely populated transient DNA local conformations is involved in processive holoenzyme formation.

Single Molecule Flourescence Methods to Monitor Site-Specific Fluctuations of Cy3 Monomer and Dimer Labeled DNA Constructs within Macromolecular Machines

Local fluctuations of the sugar-phosphate backbones of DNA (a form of DNA ‘breathing’) play key roles in protein-DNA assembly and enzymatic function. By monitoring spectroscopic signals from single-molecules of DNA constructs labeled with optical probes that are either rigidly inserted into the sugar-phosphate backbones or attached using flexible linkers, it is possible to study local conformational fluctuations within DNA at specific sites, and the dependence of these fluctuations on base sequence and secondary structure. Here we present two experimental single molecule spectroscopic methods, recently developed in our laboratory, to monitor the local fluctuations of Cy3-labeled DNA constructs. Both methods combine single-molecule total internal reflection fluorescence (TIRF) microscopy with polarized, phase-modulated optical excitation to detect, alternatively, linear and nonlinear optical signals. In Method 1 we use a linearly polarized continuous wave (cw) beam to excite the sample, so that the emitted fluorescence contains information about the rotational motion of a Cy3 monomer labeled DNA construct, or alternatively, the relative conformational changes of a Cy3 dimer inserted into the DNA sugar-phosphate backbones. In Method 2, we use sequences of broadband optical pulses to excite the sample, so that the ensuing fluorescence contains information about the linear absorbance spectrum, and alternatively, the two-dimensional fluorescence spectrum (2DFS). The latter experiments can be used to monitor the details of local conformational changes of a Cy3 dimer labeled DNA construct. Together, the two methods can be used to study biomolecular dynamics with varying levels of time resolution and structural information. For example, Method 1 can be applied to study sub-millisecond equilibrium fluctuations of Cy3 labeled DNA constructs, while Method 2 is well suited to monitor conformational transitions of a Cy3 labeled DNA construct during a biochemical reaction.

Probing (UN)Folding Transition Paths of Fast-Folding Proteins by Single-Molecule Fluorescence: Exploring the Role of Secondary Structure, Fold Topology and Sequence

Protein folding involves formation of secondary structural elements and their collapse into well-defined 3-dimensional structure. Attempts to study how these processes cooperate to form the native structure and determine folding mechanisms has been focus of research for decades. Addressing these questions require looking at the distributions of behaviors of individual molecules, which has been successfully attained by theory and computer simulations. Experimentally, the problem must be approached by single-molecule spectroscopic methods, which have not had the required time-resolution. We are investigating protein folding at the single-molecule level combining advanced custom-built fluorescence-based SM-FRET techniques optimized for high-count rates and a maximum likelihood analysis (MLA) of photon arrival times that we developed for the detection of (un)folding transition paths of microsecond folding proteins. To investigate the roles of secondary structure, fold topology, and amino acid sequence we are performing these experiments on three fast-folding protein models, all consisting of three secondary structure elements in an antiparallel arrangement: En-HD (three α-helices), and Nedd4-WW3 and FBP11WW2, which fold onto homologous 3-stranded β-sheets but with different amino acid sequence. We are investigating their folding transition paths by 2-color SM-FRET using variants labeled with FRET pairs at the two ends in free diffusion experiments. Because these proteins fold-unfold in microseconds, free diffusion offers the opportunity to catch folding transitions during the diffusive trajectories without immobilization and thus without significantly perturbing folding landscape. Our analysis of these data using MLA of photon trajectories (rather than binning) provides the free energy landscape as a function of the experimental order parameter (FRET-pair distance) and the intramolecular diffusion coefficient. Subsequent reconstruction of the molecular trajectories from the photon trajectories using stochastic methods provide a direct glimpse of barrier crossing events.

Dynamical response in methanol–chloroform binary solvent mixture over fs–μs time regime

ABSTRACTThe present study elucidates the extended nature of the weak interaction between methanol and chloroform in its mixture using steady-state, ultrafast and single molecule spectroscopic methods. UV–vis absorption spectroscopy using a solvatochromic dye indicates a synergistic solvation in the methanol–chloroform binary solvent mixture, which causes the solvatochromic dye to sense increased polarity compared to the bulk counterparts. Such synergism was not observed in the emission study and is explained by the weak nature of the interaction between methanol and chloroform. Fluorescence anisotropy and single molecule fluorescence spectroscopy are employed to understand the nature of such weak interaction on femtosecond to microsecond time scale. Retardation of both the rotational and translational diffusion of the reporter molecule indicates that the observed weak interaction is extended over large dimension in the condensed phase.

Polymer matrix dependence of conformational dynamics within a π-stacked perylenediimide dimer and trimer revealed by single molecule fluorescence spectroscopy

The conformation of embedded molecule in a polymer matrix is sensitive to the local nano-environment that the molecule experiences. Particularly, single molecule spectroscopic methods have been utilized to visualize each molecular conformation in local sites of the polymer matrix by monitoring rotational diffusion and fluctuating fluorescence of the molecule. Here, we have performed single molecule spectroscopic experiments on a π-stacked perylenediimide (PDI) dimer and trimer, in which enhanced π-π interaction in π-stacked PDIs makes the fluorescence lifetime longer, embedded in two different polymers, namely poly(methyl methacrylate) (PMMA) and poly(butyl methacrylate) (PBMA), to reveal the conformational change depending on the polymer matrix. The fluorescence lifetimes of π-stacked PDIs are influenced by polymer surroundings because their molecular conformations are dependent on their interactions with the local environment in the polymer matrix. Furthermore, from an in-depth analysis of autocorrelation functions of fluorescence intensity trajectories, we could assign that the first autocorrelation value (lag 1) is larger as the intensity trace becomes more fluctuating. Thus, we expect that π-stacked PDIs, a model system for the formation of PDI excimer-like states, can be utilized to probe the surrounding nano-environment by monitoring the conformational change in real time.

What can be learned from single molecule spectroscopy? Applications to sol–gel-derived silica materials

Single molecule spectroscopic methods are now being widely employed to probe the nanometer scale properties of sol-gel-derived silica materials. This article reviews a subset of the recent literature in this area and provides salient examples of the new information that can be obtained. The materials covered include inorganic and organically-modified silica, along with surfactant-templated mesoporous materials. Studies of molecule-matrix interactions based on ionic, hydrogen bonding and hydrophobic interactions are reviewed, highlighting the impacts of these interactions on mass transport phenomena. Quantitative investigations of molecular diffusion by single molecule tracking and fluorescence correlation spectroscopy are also covered, focusing on the characterization of anisotropic and hindered diffusion in mesoporous systems. Single molecule polarity studies are described and the new information that can be obtained from the resulting inhomogeneous distributions is discussed. Likewise, single molecule studies of silica acidity properties are reviewed, including observation of nanoscale buffering phenomena due to the chemistry of surface silanols. Finally, related single nanoparticle studies of macroporous silicas are also discussed.

Calmodulin, conformational states, and calcium signaling. A single-molecule perspective.

Single-molecule fluorescence measurements can provide a new perspective on the conformations, dynamics, and interactions of proteins. Recent examples are described illustrating the application of single-molecule fluorescence spectroscopy to calcium signaling proteins with an emphasis on the new information available in single-molecule fluorescence burst measurements, resonance energy transfer, and polarization modulation methods. Calcium signaling pathways are crucial in many cellular processes. The calcium binding protein calmodulin (CaM) serves as a molecular switch to regulate a network of calcium signaling pathways. Single-molecule spectroscopic methods can yield insights into conformations and dynamics of CaM and CaM-regulated proteins. Examples include studies of the conformations and dynamics of CaM, binding of target peptides, and interaction with the plasma-membrane Ca2+ pump. Single-molecule resonance energy transfer measurements revealed conformational substates of CaM, and single-molecule polarization modulation spectroscopy was used to probe interactions between CaM and the plasma-membrane Ca2+-ATPase.

Enhanced fluorescence of Cy5-labeled oligonucleotides near silver island films: a distance effect study using single molecule spectroscopy.

We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.

Gaining Insight into the Nanoscale Properties of Sol−Gel-Derived Silicate Thin Films by Single-Molecule Spectroscopy

The application of single-molecule spectroscopic methods in studies of individual nanoscale environments within sol-gel-derived silicate thin films is reviewed. Representative examples of the experiments performed and results obtained in several studies from the authors' laboratories are given. Included are investigations of the static and dynamic polarity properties of organically modified silicate (ORMOSIL) films. The results of these studies point to nonrandom variations in the film properties, providing strong evidence for the formation of phase-separated organic- and inorganic-rich domains. Studies of single-molecule diffusion through the same films yield important evidence for the formation of liquidlike silicate oligomers that facilitate probe molecule diffusion. Finally, single-molecule studies of the local pH within individual film environments are discussed. Valuable information on the contributions of local materials' acidity variations to overall sample heterogeneity is obtained. The results of immersion studies indicate that certain molecular environments are inaccessible to external solutions over periods as long as a few hours. The article concludes with a discussion of possible future challenges in this research that may be addressed by new and existing single-molecule methods.

Nanoscale Properties and Matrix−Dopant Interactions in Dye-Doped Organically Modified Silicate Thin Films

The nanoscale properties of organically modified sol−gel-derived silicate thin films are studied in detail by single-molecule spectroscopic methods. For these studies, the solvent-sensitive probe Nile Red is doped into the films at nanomolar concentrations. Spectroscopic data are obtained for films prepared from sols containing different mole fractions of isobutyltrimethoxysilane and tetraethoxysilane. The data are analyzed using a model based on Marcus theory, providing important new information on static local film properties such as polarity and the extent of specific dopant−matrix interactions. Data on dynamic phenomena related to local matrix rigidity is also obtained. In general, throughout the range of samples studied, the most polar environments are also found to be the most rigid. With regard to their static properties, broad heterogeneous distributions are found in films of predominantly inorganic composition. In several instances, bimodal distributions are also observed, which result from speci...

Characterization of Molecular Scale Environments in Polymer Films by Single Molecule Spectroscopy

Single molecule spectroscopic methods are used to obtain detailed information on the polarity and rigidity of molecular-scale environments found in thin poly(vinyl alcohol) (PVA) and poly(methyl methacrylate) (PMMA) films. Nile Red is employed as a highly sensitive spectroscopic probe of environmental properties in these experiments. Fluorescence spectra are recorded for numerous single molecules and their peak positions and widths determined by fitting the spectra to Gaussian functions. The spectral data are analyzed using a new model for the dependence of the Nile Red charge-transfer transition on the properties of the surrounding medium. This model is based on previous work by Marcus (Marcus, R. A. J. Phys. Chem. 1990, 94, 4963). Additional information required for the analysis is obtained from extensive bulk solution-phase absorption and fluorescence studies. A broad inhomogeneous distribution of environments is found for PVA. The results are shown to depend significantly on PVA film water content, wi...