Kinetic and vibrational isotope effects of proton transfer reactions in channelrhodopsin-2.

Channelrhodopsins (ChRs) are light-gated cation channels. After blue-light excitation, the protein undergoes a photocycle with different intermediates. Here, we have recorded transient absorbance changes of ChR2 from Chlamydomonas reinhardtii in the visible and infrared regions with nanosecond time resolution, the latter being accomplished using tunable quantum cascade lasers. Because proton transfer reactions play a key role in channel gating, we determined vibrational as well as kinetic isotope effects (VIEs and KIEs) of carboxylic groups of various key aspartic and glutamic acid residues by monitoring their C=O stretching vibrations in H2O and in D2O. D156 exhibits a substantial KIE (>2) in its deprotonation and reprotonation, which substantiates its role as the internal proton donor to the retinal Schiff base. The unusual VIE of D156, upshifted from 1736 cm(-1) to 1738 cm(-1) in D2O, was scrutinized by studying the D156E variant. The C=O stretch of E156 shifted down by 8 cm(-1) in D2O, providing evidence for the accessibility of the carboxylic group. The C=O stretching band of E90 exhibits a VIE of 9 cm(-1) and a KIE of ∼2 for the de- and the reprotonation reactions during the lifetime of the late desensitized state. The KIE of 1 determined in the time range from 20 ns to 5 ms is incompatible with early deprotonation of E90.

Pre-gating conformational changes in the ChETA variant of channelrhodopsin-2 monitored by nanosecond IR spectroscopy.

Light-gated ion permeation by channelrhodopsin-2 (ChR2) relies on the photoisomerization of the retinal chromophore and the subsequent photocycle, leading to the formation (on-gating) and decay (off-gating) of the conductive state. Here, we have analyzed the photocycle of a fast-cycling ChR2 variant (E123T mutation, also known as ChETA), by time-resolved UV/vis, step-scan FT-IR, and tunable quantum cascade laser IR spectroscopies with nanosecond resolution. Pre-gating conformational changes rise with a half-life of 200 ns, silent to UV/vis but detected by IR spectroscopy. They involve changes in the peptide backbone and in the H-bond of the side chain of the critical residue D156. Thus, the P1(500) intermediate must be separated into early and late states. Light-adapted ChR2 contains a mixture of all-trans and 13-cis retinal in a 70:30 ratio which are both photoactive. Analysis of ethylenic and fingerprint vibrations of retinal provides evidence that the 13-cis photocycle recovers in 1 ms. This recovery is faster than channel off-gating and most of the proton transfer reactions, implying that the 13-cis photocycle is of minor functional relevance for ChR2.

Monte Carlo simulation of contrast-enhanced whole brain radiotherapy on a CT scanner.

PURPOSE: To perform a feasibility study of contrast-enhanced whole brain radiotherapy for treating patients with multiple brain metastasis using a conventional computed tomography (CT) scanner. METHODS: The treatment dose was optimized to be applied in a single run using a maximum tube power of 5200 kWs at 140 kV. CT scans of a large and a small head were used as reference. Irradiation geometry, shielding, axial beam collimation, radial beam collimation, gantry tilt, and tube current for beam modulation were optimized using a Monte Carlo simulation and a contrast agent concentration of 5 mg/ml iodine in the tumor. The statistical uncertainty of the Monte Carlo simulation was corrected using back convolution. RESULTS: Using a CT tube with a beam collimation of 28.8 mm, a mean tumor dose of 1.76 +/- 0.13 Gy was achieved, while the head bone dose was 2.61 +/- 0.18 Gy with a normal brain dose of 0.98 +/- 0.06 Gy, eye dose of 0.19 +/- 0.05 Gy, and lens dose of 0.15 +/- 0.03 Gy, respectively. Using a CT tube with dose modulation and a beam collimation of 40.0 mm, the mean tumor dose was 2.00 +/- 0.11 Gy with a head bone dose of 1.96 +/- 0.14 Gy, normal brain dose of 1.13 +/- 0.08 Gy, eye dose of 0.21 +/- 0.05 Gy, and lens dose of 0.16 +/- 0.02 Gy, respectively. Thus a standard CT scanner enables an effective tumor dose of 37.0 Gy to be administered in 13 fractions, while exposing healthy brain to an effective dose of 17.2 Gy and head bone to 69.3 Gy. Additional radial collimation implemented in the hardware improves the therapeutic tumor dose by 25.2% in relation to the bone dose. CONCLUSIONS: Contrast-enhanced total brain radiotherapy is feasible using a conventional CT tube with optimized dose application.

Protein dynamics observed by tunable mid-IR quantum cascade lasers across the time range from 10ns to 1s.

We have developed a spectrometer based on tunable quantum cascade lasers (QCLs) for recording time-resolved absorption spectra of proteins in the mid-infrared range. We illustrate its performance by recording time-resolved difference spectra of bacteriorhodopsin in the carboxylic range (1800-1700cm-1) and on the CO rebinding reaction of myoglobin (1960-1840cm-1), at a spectral resolution of 1cm-1. The spectrometric setup covers the time range from 4ns to nearly a second with a response time of 10-15ns. Absorption changes as low as 1×10-4 are detected in single-shot experiments at t>1µs, and of 5×10-6 in kinetics obtained after averaging 100 shots. While previous time-resolved IR experiments have mostly been conducted on hydrated films of proteins, we demonstrate here that the brilliance of tunable quantum cascade lasers is superior to perform ns time-resolved experiments even in aqueous solution (H2O).

Kinetics of proton release and uptake by channelrhodopsin-2.

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P(3)(520) intermediate. As the P(3)(520) state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.