Tri- and pentamethine cyanine dyes for fluorescent detection of α-synuclein oligomeric aggregates.

The pathogenesis of Parkinson's disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10(-6) M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2-2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more "crescent-shape" molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.

Studies of interaction between cyanine dye T-284 and fibrillar alpha-synuclein.

A key feature of Parkinson's disease is the formation and accumulation of amyloid fibrils of the natively unfolded protein α-synuclein (ASN) inside neurons. Recently we have proposed novel sensitive monomethinecyanine dye T-284 as fluorescent probe for quantitative detection of ASN amyloid fibrils. In this study the T-284 dye complex with ASN fibril was characterized by means of fluorescence anisotropy, atomic force microscopy and time-resolved fluorescence techniques to give further insights into the mode of dye interaction with amyloid fibrils. The fluorescence anisotropy of T-284 was shown to noticeably increase upon addition of aggregated proteins indicating on stable dye/amyloid fibril complex formation. AFM imaging of fibrillar wild-type ASN revealed differences in heights between ASN fibrils alone and in presence of the T-284 dye (6.37 ± 1.0 nm and 8.0 ± 1.1 nm respectively), that is believed to be caused by embedding of T-284 dye molecules in the "binding channel" running along the fibril. Fluorescence decay analysis of the T-284 in complexes with fibrillar ASN variants revealed the fluorescence lifetime values for T-284/fibril complexes to be an order of magnitude higher as compared to the free dye. Also, the fluorescence decay of free T-284 was bi-exponential, while dye bound to protein yields tri-exponential decay. We suppose that in complexes with fibrillar ASN variants T-284 dye might exist in different "populations" due to interaction with fibrils in different conformers and ways. The exact binding mode of T-284 with ASN fibrils needs further studies. Studied parameters of dye/amyloid fibril complexes are important for the characterization and screening of newly-developed amyloid-sensitive dyes.

Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection.

We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar alpha-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The commercially available classic amyloid stain thioflavin T (Thio T) was used as the reference dye. T-284 and SH-516 dyes can be used for fluorometric quantification of fibrillar wild-type ASN at concentrations of approximately 1.5-20 microg/ml. Both dyes appeared suitable for step-wise monitoring of ASN variants (wild-type and mutants A30P and A53T) aggregation into fibrils in vitro, demonstrating good reproducibility, exceeding that for the commonly used Thio T. Our assay may be used for screening in vitro of agents capable of affecting the aggregation of ASN. In addition, T-284 and SH-516 cyanine dyes were shown to recognize amyloid proteins of various amino acid compositions selectively. T-284 demonstrated particular sensitivity to wild-type and A53T ASN, while for SH 516, the fluorescence response to fibrillar proteins was nearly the same except for lysozymes. T-284 and SH-516 cyanine dyes are sensitive and specific fluorescent probes for monitoring ASN fibril formation process in vitro, quantification of fibrillar ASN in solution, and fluorescent detection of various fibrillar protein species.

Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates.

The interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 90-mer single-strand DNA was investigated in solution by fluorescence correlation spectroscopy. Autocorrelation functions obtained in the presence of an excess of protein show larger diffusion times (tau (d)) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (theta), as a way to describe the percentage of protein bound to DNA, was directly estimated from FCS data. A significantly stronger binding capability for the cholesterol modified substrate (78% DNA bound) than for other double-stranded DNA substrates was observed, while the lowest affinity was found for the single-stranded DNA (27%). This is in accordance with a damage recognition role of the XPC protein. The similar affinity of XPC for undamaged and 'bubble' DNA substrates (58% and 55%, respectively) indicates that XPC does not specifically bind to this type of DNA substrate comprising a large (30-nt) central unpaired region.

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes.

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)).

Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.

Influence of Ca2+ cations on low pH-induced DNA structural transitions.

A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm(-1) line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm(-1)) and do not protect adenine (characteristic line at 1304 cm(-1)) and the N(7) of guanine (line at 1488 cm(-1)) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.

Real-time light-driven dynamics of the fluorescence emission in single green fluorescent protein molecules.

Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the "on-off" blinking observed in the Ser-65 --> Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 micros and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on- and off-times renders a characteristic off-time of 1.6 +/- 0.2 s and allows us to calculate a transition yield of approximately 0.5 x 10(-5) from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is approximately 1.5 kW/cm(2). On the basis of the single-molecule data we calculate an absorption cross section of 6.5 x 10(-17) cm(2) for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level.

Visualising individual green fluorescent proteins with a near field optical microscope.

The use of the green fluorescence protein (GFP) as an individual marker for applications in molecular biology requires detailed understanding of its photophysical and photodynamical properties. We investigated individual S65T mutants of GFP both on a glass surface and embedded in a water-pore gel. An aperture-type near field scanning optical microscope (NSOM) with two polarisation detection channels was applied to afford high spatial (approximately 70 nm) and temporal (0.5 ms) resolution. Shear-force and near field fluorescence imaging were performed simultaneously, allowing direct correlation between topographic and optical features. Polarisation data showed that the emission dipole moment of the proteins is fixed in space within both the barrel structure of the protein and the gel matrix used for spatial confinement of the proteins. The photophysical behaviour of the S65T-GFP mutants was monitored in time, with 500-micros real-time resolution and continuous imaging for periods of more than 2 h. Our results show the reversible on-off behaviour on a time scale that spans from 10(-4) to 10(3) s. Even a process generally identified as "bleaching" turns out to be reversible if a sufficient long observation time is allowed. As such, the photodynamics of individual GFPs appear to be much more complex than the properties deduced from ensemble-averaged measurements.

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.

Evidence for Hoogsteen GC base pairs in the proton-induced transition from right-handed to left-handed poly(dG-dC).poly(dG-dC).

The structure of double-helical poly(dG-dC).poly(dG-dC) is investigated at various pH values with Raman spectroscopy, absorption spectroscopy, and circular dichroism. A comparison is made between the B-form with Watson-Crick base pairing at 1 mM [Na+] and pH 7.2, the Z-form with Watson-Crick base pairing at 4 M [Na+] and pH 7.2, and a different structure at 1 mM [Na+] and pH 4.5 as well as at 150 mM [Na+] and pH 3.1. The CD spectrum of poly(dG-dC). poly(dG-dC) under the latter conditions does not show a negative band at 290 nm. The structure is a double-helical structure different from the B-form and the Z-form according to circular dichroism, Raman, and absorption spectroscopic studies. The Raman spectra evidence that the structure contains Hoogsteen base pairing. This can be accommodated in the double helix when the cytosine group is protonated and the sugar-guanine conformer has adopted a C2'-endo/syn conformation. It is shown that this antiparallel-stranded Hoogsteen base paired structure can be maintained under varying conditions, balancing the decrease in pH with an increased salt concentration. It is further concluded that the proton-induced transition from a Watson-Crick to a Hoogsteen base pair is aided by a decrease of [Na+] at pH 4.5 and occurs prior to a conversion from a right-handed helix to a left-handed helix.

Raman microspectroscopic approach to the study of human granulocytes.

A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

Laser irradiation and Raman spectroscopy of single living cells and chromosomes: sample degradation occurs with 514.5 nm but not with 660 nm laser light.

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot approximately 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.