Fluorescence quenching by photoinduced electron transfer: a reporter for conformational dynamics of macromolecules.

Photoinduced electron transfer (PET) between organic fluorophores and suitable electron donating moieties, for example, the amino acid tryptophan or the nucleobase guanine, can quench fluorescence upon van der Waals contact and thus report on molecular contact. PET-quenching has been used as reporter for monitoring conformational dynamics in polypeptides, proteins, and oligonucleotides. Whereas dynamic quenching transiently influences quantum yield and fluorescence lifetime of the fluorophore, static quenching in pi-stacked complexes efficiently suppresses fluorescence emission over time scales longer than the fluorescence lifetime.

Quantum dot triexciton imaging with three-dimensional subdiffraction resolution.

We describe a simple method that improves optical resolution in fluorescence microscopy approximately 1.7-fold in all three dimensions and can be implemented on any basic confocal scanning microscope. This approach is based on three-photon absorption of commercially available quantum dots generating a triple exciton (triexciton) and subsequent blue-shifted fluorescence emission following recombination of the triexciton.

Fluorescent proteins for single-molecule fluorescence applications.

We present single-molecule fluorescence data of fluorescent proteins GFP, YFP, DsRed, and mCherry, a new derivative of DsRed. Ensemble and single-molecule fluorescence experiments proved mCherry as an ideally suited fluorophore for single-molecule applications, demonstrated by high photostability and rare fluorescence-intensity fluctuations. Although mCherry exhibits the lowest fluorescence quantum yield among the fluorescent proteins investigated, its superior photophysical characteristics suggest mCherry as an ideal alternative in single-molecule fluorescence experiments.

Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging.

We introduce a general approach for multicolor subdiffraction-resolution fluorescence imaging based on photoswitching of standard organic fluorophores. Photoswitching of ordinary fluorophores such as ATTO520, ATTO565, ATTO655, ATTO680, or ATTO700, i.e.

Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores.

Understanding the structural organization of biomolecules in cells, sub-cellular compartments or membranes requires non-invasive methods of observation that provide high spatial resolution. Recent advancements in fluorescence microscopy paved the way for novel super-resolution observations with an optical resolution well below the diffraction barrier of light. Here, we demonstrate that commercially available standard fluorescent probes, i.

Changes in conformational dynamics of mRNA upon AtGRP7 binding studied by fluorescence correlation spectroscopy.

The clock-regulated RNA recognition motif (RRM)-containing protein AtGRP7 (Arabidopsis thaliana glycine-rich RNA-binding protein) influences the amplitude of its transcript oscillation at the post-transcriptional level. This autoregulation relies on AtGRP7 binding to its own pre-mRNA. The sequence and structural requirements for this interaction are unknown at present.

Organelle-specific isoenzymes of plant V-ATPase as revealed by in vivo-FRET analysis.

Background: The V-ATPase (VHA) is a protein complex of 13 different VHA-subunits. It functions as an ATP driven rotary-motor that electrogenically translocates H+ into endomembrane compartments. In Arabidopsis thaliana V-ATPase is encoded by 23 genes posing the question of specific versus redundant function of multigene encoded isoforms.

C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity.

The p53 family is known as a family of transcription factors with functions in tumor suppression and development. Whereas the central DNA-binding domain is highly conserved among the three family members p53, p63 and p73, the C-terminal domains (CTDs) are diverse and subject to alternative splicing and post-translational modification. Here we demonstrate that the CTDs strongly influence DNA binding and transcriptional activity: while p53 and the p73 isoform p73gamma have basic CTDs and form weak sequence-specific protein-DNA complexes, the major p73 isoforms have neutral CTDs and bind DNA strongly.

p73 poses a barrier to malignant transformation by limiting anchorage-independent growth.

p53 is known to prevent tumour formation by restricting the proliferation of damaged or oncogene-expressing cells. In contrast, how the p53 family member p73 suppresses tumour formation remains elusive. Using a step-wise transformation protocol for human cells, we show that, in premalignant stages, expression of the transactivation-competent p73 isoform TAp73 is triggered in response to pRB pathway alterations.

Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis.

Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips.

Probing polyproline structure and dynamics by photoinduced electron transfer provides evidence for deviations from a regular polyproline type II helix.

Polyprolines are well known for adopting a regular polyproline type II helix in aqueous solution, rendering them a popular standard as molecular ruler in structural molecular biology. However, single-molecule spectroscopy studies based on Förster resonance energy transfer (FRET) have revealed deviations of experimentally observed end-to-end distances of polyprolines from theoretical predictions, and it was proposed that the discrepancy resulted from dynamic flexibility of the polyproline helix. Here, we probe end-to-end distances and conformational dynamics of poly-l-prolines with 1-10 residues using fluorescence quenching by photoinduced-electron transfer (PET).

A highly sensitive particle agglutination assay for the detection of P53 autoantibodies in patients with lung cancer.

Background: Numerous assays have been described for the detection of p53 autoantibodies. These assays are highly specific with low sensitivity. In this report, the authors describe a highly sensitive and simple particle agglutination immunoassay using superparamagnetic particles for capturing p5 autoantibodies, p53 protein, and p53 protein-antibody complexes from large volumes of serum samples (2 mL).

Single-molecule fluorescence resonance energy transfer in nanopipets: improving distance resolution and concentration range.

In recent years fluorescence resonance energy transfer (FRET) has widely been used to measure distances, binding, and distance dynamics at the single-molecule (sm) level. Some basic constraints of smFRET are the limited distance resolution owing to low photon statistics and the restriction to high affinity interactions. We demonstrate that by confining molecules in nanopipets with an inner diameter of approximately 100 nm at the tip, FRET can be measured with improved photon statistics and at up to 50-fold higher concentrations.

Fluorescence of single molecules in polymer films: sensitivity of blinking to local environment.

The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed.

Polymer properties of polythymine as revealed by translational diffusion.

Biopolymers, such as single-stranded DNA (ssDNA), are often described as semiflexible polymers or wormlike chains. We investigated the length dependence of diffusional properties of homogeneous ssDNA (polythymine) with up to 100 nucleotides using fluorescence correlation spectroscopy. We found that the hydrodynamic radius Rh scales according to a power law, with an exponent between 0.

Chemical and biological investigations of beta-oligoarginines.

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1).

DNA-based molecular wires: multiple emission pathways of individual constructs.

The extent of photon energy transfer through individual DNA-based molecular wires composed of five dyes is investigated at the single molecular level. Combining single-molecule spectroscopy and pulse interleaved excitation imaging, we have directly resolved the time evolution spectral response of individual constructs, while simultaneously probing DNA integrity. Our data clearly show that intact wires exhibit photon-transfer efficiencies close to 100% across five dyes.

Dissecting and reducing the heterogeneity of excited-state energy transport in DNA-based photonic wires.

Molecular photonic wires are one-dimensional representatives of a family of nanoscale molecular devices that transport excited-state energy over considerable distances in analogy to optical waveguides in the far-field. In particular, the design and synthesis of such complex supramolecular devices is challenging concerning the desired homogeneity of energy transport. On the other hand, novel optical techniques are available that permit direct investigation of heterogeneity by studying one device at a time.

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy.

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes.

p53 family members in myogenic differentiation and rhabdomyosarcoma development.

The p53 family comprises the tumor suppressor p53 and the structural homologs p63 and p73. How the three family members cooperate in tumor suppression remains unclear. Here, we report different but complementary functions of the individual members for regulating retinoblastoma protein (RB) function during myogenic differentiation.

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy.

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5' end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity.

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy.

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes.

Radiative and nonradiative rate fluctuations of single colloidal semiconductor nanocrystals.

Spectrally and time-resolved single-molecule fluorescence spectroscopy was used to investigate fluctuations of the photophysical characteristics of different types of semiconductor nanocrystals (NCs) at room temperature. Correlation of photoluminescence (PL) emission maxima, decay time, and intensity of individual NCs with millisecond time resolution reveals new sources of intensity fluctuations and photophysical properties. In particular, we demonstrate that independent of quenched states spectral diffusion is associated with changes of the radiative rate constant k(r) by means of the quantum-confined Stark effect.