Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed
It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA-Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA-Cer with Myr-Pal-YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal-YFP exhibited a much faster mobility compared with HA-Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.
Double‐stranded DNA offers multiple binding sites to DNA stains. Measurements of noncovalently bound dye–nucleic acid complexes are, necessarily, measurements of an ensemble of chromophores. Thus, it is difficult to assign fluorescence properties to base‐pair‐specific binding modes of cyanine dyes or, vice versa, to obtain information about the local environment of cyanines in nucleic acids by using optical spectroscopy. The feasibility to stain DNA and simultaneously probe local perturbations by optical spectroscopy would be a valuable asset to nucleic acid research. So‐called FIT probes (forced intercalation probes) were used to pinpoint the location of the DNA stain thiazole orange (TO) in PNA⋅DNA duplexes. A detailed analysis of the base‐pair dependence of optical properties is provided and enforced binding of TO is compared with “classical” binding of free TO‐PRO1. UV‐visible absorbance, circular dichroism (CD) and fluorescence spectroscopy, and melting‐curve analyses confirmed site‐specific TO intercalation. Thiazole orange exhibited base‐specific responses that are not observed in noncovalent dye–nucleic acid complexes, such as an extraordinary dependence of the TO extinction coefficient (±60 % variation of the averaged εmax of 57 000 M−1 cm−1) on nearest‐neighbor base pairs. TO signals hybridization, as shown by increases in the steady‐state fluorescence emission. Studies of TO fluorescence lifetimes in FIT–PNA and in DNA⋅DNA and PNA⋅DNA complexes highlighted four different fluorescence‐decay processes that may be closed or opened in response to matched or single‐mismatched hybridization. A very fast decay process (0.04–0.07 ns) and a slow decay process (2.33–3.95 ns) provide reliable monitors of hybridization, and the opening of a fast decay channel (0.22–0.48 ns) that resulted in an attenuation of the fluorescence emission is observed upon the formation of mismatched base pairs.
Experimental realizations of two-dimensional (2D) electronic spectroscopy in the ultraviolet (UV) must so far contend with a limited bandwidth in both the excitation and particularly the probe frequency. The pump bandwidth is at best 1500 cm −1 (full width at half maximum) at a fixed wavelength of 267 nm or 400 cm −1 for tunable pulses. The use of a replica of the pump pulse as a probe limits the observation of photochemical processes to the excitation region and makes the disentanglement of overlapping signal contributions difficult. We show that 2D Fourier transform spectroscopy can be conducted in a shaper-assisted collinear setup comprising fully tunable UV pulse pairs and supercontinuum probe spanning 250-720 nm. The pump pulses are broadened up to a useable spectral coverage of 2000 cm −1 (25 nm at 316 nm) by self-phase modulation in bulk CaF 2 and compressed to 18 fs. By referencing the white light probe and eliminating pump stray light contributions, high signal-to-noise ratios even for weak probe intensities are achieved. Data acquisition times as short as 4 min for a selected population time allow the rapid recording of 2D spectra for photolabile biological samples even with the
Direct pulse shaping in the UV was used to compress and structure pulses throughout the range of 250 - 400 nm. Broadband pulses generated by SHG of a NOPA were used as input to an acousto-optic programmable dispersive filter. As this shaper creates lateral dispersion, aspects of Gaussian and geometric optics had to be considered for the design of the beam path. Special care was taken to produce a homogeneous input beam. We show nearly Fourier-limited pulses as short as 16.8 fs at 320 nm and 19.5 fs at 260 nm. Full control over amplitude and phase is demonstrated by generating arbitrary shapes like square pulses and complex pulse sequences. The subpulses were manipulated individually in intensity, temporal delay, chirp, relative phase and central wavelength.
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