Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.     

Probing the ATP-induced conformational flexibility of the PcrA helicase protein using molecular dynamics simulation

Helicases are enzymes that unwind double-stranded DNA (dsDNA) into its single-stranded components. It is important to understand the binding and unbinding of ATP from the active sites of helicases, as this knowledge can be used to elucidate the functionality of helicases during the unwinding of dsDNA. In this work, we investigated the unbinding of ATP and its effect on the active-site residues of the helicase PcrA using molecular dynamic simulations. To mimic the unbinding process of ATP from the active site of the helicase, we simulated the application of an external force that pulls ATP from the active site and computed the free-energy change during this process. We estimated an energy cost of ~85 kJ/mol for the transformation of the helicase from the ATP-bound state (1QHH) to the ATP-free state (1PJR). Unbinding led to conformational changes in the residues of the protein at the active site. Some of the residues at the ATP-binding site were significantly reoriented when the ATP was pulled. We observed a clear competition between reorientation of the residues and energy stabilization by hydrogen bonds between the ATP and active-site residues. We also checked the flexibility of the PcrA protein using a principal component analysis of domain motion. We found that the ATP-free state of the helicase is more flexible than the ATP-bound state.     

Sequence dependence of DNA conformational flexibility

By using conformational free energy calculations, we have studied the sequence dependence of flexibility and its anisotropy along various conformational variables of DNA base pairs. The results show the AT base step to be very flexible along the twist coordinate. On the other hand, homonucleotide steps, GG(CC) and AA(TT), are among the most rigid sequences. For the roll motion that would correspond to a bend, the TA step is most flexible, while the GG(CC) step is least flexible. The flexibility of roll is quite anisotropic; the ratio of fluctuations toward the major and minor grooves is the largest for the GC step and the smallest for the AA(TT) and CG steps. Propeller twisting of base pairs is quite flexible, especially of A.T base pairs; propeller twist can reach 19 degrees by thermal fluctuation. We discuss the effect of electrostatic parameters, comparison with available experimental results, and biological relevance of these results.     

Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy

Prior to undergoing postsynthetic 3'-5' editing (proofreading), a defective DNA primer terminus must be transferred from the 5'-3' polymerase active site to a remote 3'-5' exonuclease site. To elucidate the mechanisms by which this occurs, we have used time-resolved fluorescence spectroscopy to study the interaction of dansyl-labeled DNA primer/templates with the Klenow fragment of Escherichia coli DNA polymerase I. The dansyl probe is positioned such that when the DNA substrate occupies the polymerase active site, the probe is solvent-exposed and possesses a short average fluorescence lifetime (4.7 ns) and extensive angular diffusion (42.5 degrees). Conversely, when the DNA substrate occupies the exonuclease active site, the probe becomes buried within the protein, resulting in an increase in the average lifetime (14.1 ns) and a decrease in the degree of angular diffusion (14.4 degrees ). If both polymerase and exonuclease binding modes are populated (lower limit approximately 5%), their markedly different fluorescence properties cause the anisotropy to decay with a characteristic "dip and rise" shape. Nonlinear least-squares analysis of these data recovers the ground-state mole fractions of exposed (x(e)) and buried (x(b)) probes, which are equivalent to the equilibrium proportions of the DNA substrate bound at the polymerase and exonuclease sites, respectively. The distribution between the polymerase and exonuclease binding modes is given by the equilibrium partitioning constant K(pe) (equal to x(b)/x(e)). The important determinants of the proofreading process can therefore be identified by changes made to either the protein or DNA that perturb the partitioning equilibrium and hence alter the magnitude of K(pe).     

Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: a photon and fluorescence correlation spectroscopy study

Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010. The apparent dissociation constants, Kd(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5'-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, respectively. Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity. At pH 7.6 in the presence of ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM. The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA. Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R(h), of hRepA dimer and its complex with ssDNA as determined by FCS (R(h) = 6.6 +/- 1 nm). hRepA starts to aggregate under acidic conditions (相似文献   

Probing the conformational switch of I-motif DNA using tunable resistive pulse sensing

I-motif DNA, which can fold and unfold reversibly in various environments, plays a significant role in DNA nanotechnology and biological functions. Thus, it is of fundamental importance to identify the different conformations of i-motif DNA. Here, we demonstrate that distinct structures of i-motif DNA conjugated to polystyrene spheres can be distinguished through tunable resistive pulse sensing technique. When dispersed in acidic buffer, i-motif DNA coating on polystyrene spheres would fold into quadruplex structure and subsequently induce an apparent increase in the translocation duration time upon passing through a nanopore due to the shielding effect of the surface charge of the nanospheres. However, if the DNA strands don't have conformational changes in acidic buffer, little shift can be observed in the translocation duration time of the DNA functionalized polystyrene spheres. A before-and-after assay was also performed to illustrate the fast speed of i-motif DNA folding using this technique. The successful implementation of tunable resistive pulse sensing to monitor the conformational transition of i-motif DNA provides a potential tool to detect the structural changes of DNA and an alternative approach to study the function of DNA structures.     

DNA length evaluation using cyanine dye and fluorescence correlation spectroscopy

To develop a high-performance method for measuring the length of double-stranded DNA (dsDNA) fragments, the capability of fluorescence correlation spectroscopy (FCS) was examined. To omit troublesome and time-consuming labeling operations such as PCR with fluorescently labeled mononucleotides or primers, intercalation of dimeric cyanine dye YOYO-1 iodide (YOYO) to dsDNA was utilized as a simple labeling method. Various lengths of dsDNA fragments were prepared and mixed with YOYO prior to FCS, and the dependence of the diffusion time of a dsDNA-YOYO complex on the length of dsDNA fragment and the dsDNA/YOYO ratio was investigated. It was successfully demonstrated that the dsDNA length can be measured using YOYO and FCS, and the calibration curve was developed taking into account the rewinding and expansion of the dsDNA fragment caused by YOYO intercalation.     

Probing conformational changes of proteins by quantitative second-derivative infrared spectroscopy

Probing protein conformational changes plays a crucial role in protein structure and function studies. However, the lack of efficient biophysical techniques makes it difficult to obtain the distinct behaviors of different secondary structure elements in a protein upon perturbation. This paper presents a discussion of the two major problems, the effect of sidelobes and different half-width at half-height (HWHH) values, encountered in quantitative second-derivative infrared (QSD-IR) spectroscopy and introduces the development of two criteria for checking the validity of the results obtained using the QSD-IR method. It was found that neither the sidelobes nor the HWHH significantly affected the quantitative result of protein conformational changes by using poly-l-lysine and hemoglobin as model proteins. A case study of bovine serum albumin (BSA) thermal aggregation suggested that the thermal transition of BSA was a process involving sequential events, and the two helical components were found to have a distinct response to heat perturbation. These results were confirmed by two-dimensional infrared correlation spectroscopy and by results in literature, suggesting that the QSD-IR method might be a potentially powerful tool to probe the distinct response of different secondary structures to perturbation.     

Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy

We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g(0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.     

Probing the conformational heterogeneity of the acetylaminofluorene-modified 2'-deoxyguanosine and DNA by 19F NMR spectroscopy.

19F NMR spectroscopy was used to probe the conformation of a DNA adduct derived from the carcinogen 7-fluoro-N-acetyl-2-aminofluorene (FAAF) in three structural contexts: as a monomer and incorporated into single- and double-stranded DNA. The 19F NMR spectrum of dG-C8-FAAF [N-(deoxyguanosin-8-yl)-N-acetyl-7-fluoro-2-aminofluorene] in methanol at -30 degrees C exhibited four interconvertible signals in a 11:52:26:11 ratio. Dynamic NMR analysis indicated that the four torsional isomers arise from restricted rotation about the amide (gamma) (14.4 kcal/mol) and the guanyl-nitrogen (alpha) bonds. The conformational heterogeneity persisted in a single strand FAAF-12-mer, d(CTTCTTG[FAAF]ACCTC), whose 19F NMR spectrum at 22 degrees C and pH 7.0 gave only two signals in a 40:60 ratio, instead of four. The two 19F signals followed a two-site exchange with the rotation barrier of 14.7 kcal/mol about the amide (gamma') bond. A similar conformational theme was observed in the FAAF-12-mer duplex, d(CTTCTTG[FAAF]ACCTC).d(GAGGTCAAGAAG), which revealed two 19F resonances in a 41:59 ratio at 22 degrees C and pH 7.0. According to solvent-induced isotope and magnetic anisotropy effects, the two duplex conformers adopt exclusively a base displacement structure, being different only in their relative acetyl group orientations, cis (gamma' approximately 180 degrees) or trans (gamma' approximately 0 degrees ). Dynamic NMR data indicated that the two conformers do not exchange over a wide range of temperatures. This contrasts with the nonacetylated counterpart, which exhibits an equilibrium between the "B-type" and "stacked" conformers [Zhou, L., et al. (1997) J. Am. Chem. Soc. 119, 5384-5389]. The exclusive stacked nature of the AAF adducts may provide insight into why AAF adducts are more mutagenic and prone to repair than the nonacetylated AF adducts.     

Probing DNA clamps with single-molecule force spectroscopy

Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.     

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.     

Detection of specific DNA sequences using dual-color two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a biomedical research tool. FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. They can also serve as a diagnostic, detecting particular molecular species with high sensitivity and specificity. We here demonstrate that dual-color FCS measurements can be applied to detect and quantify the concentration of specific non-fluorescent molecular species without requiring any modifications to the molecule of interest. We demonstrate this capability by applying dual-color two-photon fluorescence cross-correlation spectroscopy to detect single stranded gamma tubulin DNA in solution with high sensitivity. This quantification is independent of molecular size, and the methods introduced can be extended to measurements in complex environments such as within living cells.     

Measuring conformational dynamics of biomolecules by single molecule fluorescence spectroscopy

Dynamic structural changes of macromolecules undergoing biochemical reactions can be studied using novel single molecule spectroscopy tools. Recent advances in applying such distance and orientation molecular rulers to biological systems are reviewed, and future prospects and challenges are discussed.     

Probing conformational changes of gramicidin ion channels by single-molecule patch-clamp fluorescence microscopy

Complex conformational changes influence and regulate the dynamics of ion channels. Such conformational changes are stochastic and often inhomogeneous, which makes it extremely difficult, if not impossible, to characterize them by ensemble-averaged experiments or by single-channel recordings of the electric current that report the open-closed events but do not specifically probe the associated conformational changes. Here, we report our studies on ion channel conformational changes using a new approach, patch-clamp fluorescence microscopy, which simultaneously combines single-molecule fluorescence spectroscopy and single-channel current recordings to probe the open-closed transitions and the conformational dynamics of individual ion channels. We demonstrate patch-clamp fluorescence microscopy by measuring gramicidin ion channel conformational changes in a lipid bilayer formed at a patch-clamp micropipette tip under a buffer solution. By measuring single-pair fluorescence resonance energy transfer and fluorescence self-quenching from dye-labeled gramicidin channels, we observed that the efficiency of single-pair fluorescence resonance energy transfer and self-quenching is widely distributed, which reflects a broad distribution of conformations. Our results strongly suggest a hitherto undetectable correlation between the multiple conformational states of the gramicidin channel and its closed and open states in a lipid bilayer.     

Probing protein-surfactant interaction by steady state and time-resolved fluorescence spectroscopy

The microenvironment of the probe coumarin 153 (C-153) in 1% bovine serum albumin (BSA) is more hydrophobic in nature compared to that in pure micelles or protein-surfactant complexes. In the native state of protein, we have not observed any solvation using C-153 as a probe but we have observed a slow dynamics on protein surface using 8-anilino-1-naphthalenesulfonic acid (ANS) as a probe. This may be due to the location of the probe (C-153) in the hydrophobic, solvent-inaccessible pocket of the BSA. Solvation dynamics in the BSA-surfactant (SDS) complexes in the solution phase is markedly different from that in pure micelles. This is may be due to the formation of 'necklace and bead' structure in the complexes. The rotational motion is also severely hindered in the surface of the protein.     

Exploring rare conformational species and ionic effects in DNA Holliday junctions using single-molecule spectroscopy

The four-way DNA (Holliday) junction is an essential intermediate in DNA recombination, and its dynamic characteristics are likely to be important in its cellular processing. In our previous study we observed transitions between two antiparallel stacked conformations using a single-molecule fluorescence approach. The magnesium concentration-dependent rates of transitions between stacking conformers suggested that an unstacked open structure, which is stable in the absence of metal ions, is an intermediate. Here, we sought to detect possible rare species such as open and parallel conformations and further characterized ionic effects. The hypothesized open intermediate cannot be resolved directly due to the limited time resolution and sensitivity, but our study suggests that the open form is achieved very frequently, hundreds of times per second under physiologically relevant conditions. Therefore despite being a minority species, its frequent formation raises the probability that it could become stabilized by protein binding. By contrast, we cannot detect even a transient existence of the junctions in a parallel form, and the probability of such forms with a lifetime greater than 5 ms is less than 0.01%. Stacking conformer transitions are observable in the presence of sodium or hexammine cobalt (III) ions as well as magnesium ions, but the transition rates are higher for lower valence ions at the same concentrations. This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms.