Light-Induced TripletTriplet Electron Resonance Spectroscopy

We present a new technique, light-induced triplet-triplet electron resonance spectroscopy (LITTER), which measures the dipolar interaction between two photoexcited triplet states, enabling both the distance and angular distributions between the two triplet moieties to be determined on a nanometer scale. This is demonstrated for a model bis-porphyrin peptide that renders dipolar traces with strong orientation selection effects. Using simulations and density functional theory calculations, we extract distance distributions and relative orientations of the porphyrin moieties, allowing the dominant conformation of the peptide in a frozen solution to be identified. LITTER removes the requirement of current light-induced electron spin resonance pulse dipolar spectroscopy techniques to have a permanent paramagnetic moiety, becoming more suitable for in-cell applications and facilitating access to distance determination in unmodified macromolecular systems containing photoexcitable moieties. LITTER also has the potential to enable direct comparison with Förster resonance energy transfer and combination with microscopy inside cells

Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical- Pairs

Cryptochromes (Cry) have been suggested to form the basis of light-dependent magnetic compass orientation in birds. However, to function as magnetic compass sensors, the cryptochromes of migratory birds must possess a number of key biophysical characteristics. Most importantly, absorption of blue light must produce radical pairs with lifetimes longer than about a microsecond. Cryptochrome 1a (gwCry1a) and the photolyase-homology-region of Cry1 (gwCry1-PHR) from the migratory garden warbler were recombinantly expressed and purified from a baculovirus/Sf9 cell expression system. Transient absorption measurements show that these flavoproteins are indeed excited by light in the blue spectral range leading to the formation of radicals with millisecond lifetimes. These biophysical characteristics suggest that gwCry1a is ideally suited as a primary light-mediated, radical-pair-based magnetic compass recepto

Singlet-triplet dephasing in radical pairs in avian cryptochromes leads to time-dependent magnetic field effects

Cryptochrome 4a (Cry4a) has been proposed as the sensor at the heart of the magnetic compass in migratory songbirds. Blue-light excitation of this protein produces magnetically sensitive flavin–tryptophan radical pairs whose properties suggest that Cry4a could indeed be suitable as a magnetoreceptor. Here, we use cavity ring-down spectroscopy to measure magnetic field effects on the kinetics of these radical pairs in modified Cry4a proteins from the migratory European robin and from nonmigratory pigeon and chicken. B1/2, a parameter that characterizes the magnetic field-dependence of the reactions, was found to be larger than expected on the basis of hyperfine interactions and to increase with the delay between pump and probe laser pulses. Semiclassical spin dynamics simulations show that this behavior is consistent with a singlet–triplet dephasing (STD) relaxation mechanism. Analysis of the experimental data gives dephasing rate constants, rSTD, in the range 3–6 × 107 s −1 . A simple “toy” model due to Maeda, Miura, and Arai [Mol. Phys. 104, 1779–1788 (2006)] is used to shed light on the origin of the time-dependence and the nature of the STD mechanism. Under the conditions of the experiments, STD results in an exponential approach to spin equilibrium at a rate considerably slower than rSTD. We attribute the loss of singlet–triplet coherence to electron hopping between the second and third tryptophans of the electron transfer chain and comment on whether this process could explain differences in the magnetic sensitivity of robin, chicken, and pigeon Cry4a’s

Chemical Magnetoreception: Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical-Pairs

Cryptochromes (Cry) have been suggested to form the basis of light-dependent magnetic compass orientation in birds. However, to function as magnetic compass sensors, the cryptochromes of migratory birds must possess a number of key biophysical characteristics. Most importantly, absorption of blue light must produce radical pairs with lifetimes longer than about a microsecond. Cryptochrome 1a (gwCry1a) and the photolyase-homology-region of Cry1 (gwCry1-PHR) from the migratory garden warbler were recombinantly expressed and purified from a baculovirus/Sf9 cell expression system. Transient absorption measurements show that these flavoproteins are indeed excited by light in the blue spectral range leading to the formation of radicals with millisecond lifetimes. These biophysical characteristics suggest that gwCry1a is ideally suited as a primary light-mediated, radical-pair-based magnetic compass receptor

Broadband cavity-enhanced detection of magnetic field effects in chemical models of a cryptochrome magnetoreceptor

Broadband cavity-enhanced absorption spectroscopy (BBCEAS) is shown to be a sensitive method for the detection of magnetic field effects (MFEs) in two flavin-based chemical reactions which are simple models for cryptochrome magnetoreceptors. The advantages of optical cavity-based detection and (pseudo-white-light) supercontinuum radiation have been combined to provide full spectral coverage across the whole of the visible spectrum (425 < λ < 700 nm). This region covers the absorbance spectra of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as well as their photogenerated radicals. To illustrate the power of this technique, BBCEAS has been used to record the spectral dependence of MFEs for photoinduced radical pairs formed in the intermolecular reaction of FMN with lysozyme and the intramolecular photochemistry of FAD. These reactions have been chosen for their photochemical similarities to cryptochrome proteins which have been proposed as key to the magnetic compass sense of many animals including birds. In experiments performed using low protein concentrations (10 μM) and 1 mm optical path-lengths, absorbance changes as small as 1 × 10(-7) (representing <0.1% MFEs) have been detected with good signal-to-noise offering the prospect of sensitive MFE detection in cryptochrome

Broadband cavity-enhanced detection of magnetic field effects in chemical models of a cryptochrome magnetoreceptor

Broadband cavity-enhanced absorption spectroscopy (BBCEAS) is shown to be a sensitive method for the detection of magnetic field effects (MFEs) in two flavin-based chemical reactions which are simple models for cryptochrome magnetoreceptors. The advantages of optical cavity-based detection and (pseudo-white-light) supercontinuum radiation have been combined to provide full spectral coverage across the whole of the visible spectrum (425 < λ < 700 nm). This region covers the absorbance spectra of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) as well as their photogenerated radicals. To illustrate the power of this technique, BBCEAS has been used to record the spectral dependence of MFEs for photoinduced radical pairs formed in the intermolecular reaction of FMN with lysozyme and the intramolecular photochemistry of FAD. These reactions have been chosen for their photochemical similarities to cryptochrome proteins which have been proposed as key to the magnetic compass sense of many animals including birds. In experiments performed using low protein concentrations (10 μM) and 1 mm optical path-lengths, absorbance changes as small as 1 × 10(-7) (representing <0.1% MFEs) have been detected with good signal-to-noise offering the prospect of sensitive MFE detection in cryptochrome

Effect of magnetic fields on cryptochrome-dependent responses in Arabidopsis thaliana

The scientific literature describing the effects of weak magnetic fields on living systems contains a plethora of contradictory reports, few successful independent replication studies and a dearth of plausible biophysical interaction mechanisms. Most such investigations have been unsystematic, devoid of testable theoretical predictions and, ultimately, unconvincing. A recent study, of magnetic responses in the model plant Arabidopsis thaliana, however, stands out; it has a clear hypothesis-that seedling growth is magnetically sensitive as a result of photoinduced radical-pair reactions in cryptochrome photoreceptors-tested by measuring several cryptochrome-dependent responses, all of which proved to be enhanced in a magnetic field of intensity 500 muT. The potential importance of this study in the debate on putative effects of extremely low-frequency electromagnetic fields on human health prompted us to subject it to the 'gold standard' of independent replication. With experimental conditions chosen to match those of the original study, we have measured hypocotyl lengths and anthocyanin accumulation for Arabidopsis seedlings grown in a 500 microT magnetic field, with simultaneous control experiments at 50 microT. Additionally, we have determined hypocotyl lengths of plants grown in 50 microT, 1 mT and approximately 100 mT magnetic fields (with zero-field controls), measured gene (CHS, HY5 and GST) expression levels, investigated blue-light intensity effects and explored the influence of sucrose in the growth medium. In no case were consistent, statistically significant magnetic field responses detecte

Light-Induced Triplet-Triplet Electron Resonance Spectroscopy

We present a new technique, light-induced triplet-triplet electron resonance spectroscopy (LITTER), which measures the dipolar interaction between two photoexcited triplet states, enabling both the distance and angular distributions between the two triplet moieties to be determined on a nanometer scale. This is demonstrated for a model bis-porphyrin peptide that renders dipolar traces with strong orientation selection effects. Using simulations and density functional theory calculations, we extract distance distributions and relative orientations of the porphyrin moieties, allowing the dominant conformation of the peptide in a frozen solution to be identified. LITTER removes the requirement of current light-induced electron spin resonance pulse dipolar spectroscopy techniques to have a permanent paramagnetic moiety, becoming more suitable for in-cell applications and facilitating access to distance determination in unmodified macromolecular systems containing photoexcitable moieties. LITTER also has the potential to enable direct comparison with F\uf6rster resonance energy transfer and combination with microscopy inside cells