Initial Reaction Dynamics of Proteorhodopsin Observed by Femtosecond Infrared and Visible Spectroscopy

Abstract

We present a comparative study using femtosecond pump/probe spectroscopy in the visible and infrared of the early photodynamics of solubilized proteorhodopsin (green absorbing variant) in D(2)O with deprotonated (pD 9.2) and protonated (pD 6.4) primary proton acceptor Asp-97. The vis-pump/vis-probe experiments show a kinetic isotope effect that is more pronounced for alkaline conditions, thus decreasing the previously reported pH-dependence of the primary reaction of proteorhodopsin in H(2)O. This points to a pH dependent H-bonding network in the binding pocket of proteorhodopsin, that directly influences the primary photo-induced dynamics. The vis-pump/IR-probe experiments were carried out in two different spectral regions and allowed to monitor the retinal C=C (1500 cm(-1)-1580 cm(-1)) and C=N stretching vibration as well as the amide I mode of the protein (1590 cm(-1)-1680 cm(-1)). Like the FTIR spectra of the K intermediate (PR(K)-PR difference spectra) in this spectral range, the kinetic parameters and also the quantum efficiency of photo-intermediate formation are found to be virtually independent of the pD value.

Molecular Mechanism of Uncaging CO2 from Nitrophenylacetate Provides General Guidelines for Improved ortho‐Nitrobenzyl Cages

“…[14] The experimental setup for femtosecond IR spectroscopy has been described elsewhere. [15] Excitation pulses were generated by either third harmonic generation (258 nm) or sum frequency mixing (290 nm) of tunable pulses generated with a noncollinear optical parametric amplifier and laser fundamental pulses. Large sample volumes (200 mL, c = 20 mm) were continuously stirred and pumped through a flow cell (d = 50 mm) for sufficient sample exchange.…”

Photoresponsive Formation of an Intermolecular Minimal G‐Quadruplex Motif

“…The azobenzene isomerization and the subsequent disruption of the G-quadruplex structure led to ab athochromic shift (1675 cm À1 !1667 cm À1 )o ft he C=Os tretching band [39][40][41] and to batho-and hypsochromic shifts of the bands associated with the C = Ca nd C = Np urine ring vibrations (1550-1590 cm À1 range), [39,41] features reported in the literature to be characteristic of G-quadruplex melting. [39,41] Ultrafast UVpump/mid-IR-probe experiments [42] in the carbonyl stretching vibration range (Figure 3j,t op left;F igure S54-55) were performed to investigate the dynamics of the G-quadruplex after E azobenzene excitation (l = 335 nm). In the first picoseconds after laser excitation, the transient absorption data are dominated by the cooling dynamics of the C = O stretching band (1645-1680 cm À1 )w ith al ifetime of circa 11 ps.T he absorption and bleach bands for the product state become visible after circa 10 ps (see the infinity spectrum in Figure 3j,t op right).…”

The Photocycle of Channelrhodopsin‐2: Ultrafast Reaction Dynamics and Subsequent Reaction Steps

“…[25,26] In transient absorption measurements of PR this is reflected by a wavelength-dependent delay of the stimulated emission. [25,27] In many transient absorption experiments of BR this delay is not observed. [14,28,29] Instead, the analysis of both the stimulated emission [16] and the fluorescence dynamics at the high-energy side of the band revealed a decay component in the range of 100-200 fs.…”