ABSTRACT
Homo-dimer formation is important for the function of many proteins. Although dimeric forms of cryptochromes (Cry) have been found by crystallography and were recently observed in vitro for European robin Cry4a, little is known about the dimerization of avian Crys and the role it could play in the mechanism of magnetic sensing in migratory birds. Here, we present a combined experimental and computational investigation of the dimerization of robin Cry4a resulting from covalent and non-covalent interactions. Experimental studies using native mass spectrometry, mass spectrometric analysis of disulfide bonds, chemical cross-linking, and photometric measurements show that disulfide-linked dimers are routinely formed, that their formation is promoted by exposure to blue light, and that the most likely cysteines are C317 and C412. Computational modeling and molecular dynamics simulations were used to generate and assess a number of possible dimer structures. The relevance of these findings to the proposed role of Cry4a in avian magnetoreception is discussed.
Subject(s)
Cryptochromes , Songbirds , Animals , Cryptochromes/chemistry , Dimerization , Songbirds/metabolism , LightABSTRACT
We present time-resolved optical absorption and magnetic field effect data on the photochemistry following blue light excitation of flavin adenine dinucleotide (FAD) in aqueous solution in the pH range 2.3 to 8.0. Effects of closed form conformations of FAD in ground, excited singlet, and radical pair states exhibit significant influence on the observed kinetics and magnetic field dependence and remarkably, magnetic field effects are observed even at physiological pH where the FAD radical pairs are only 75% less magnetic field sensitive than at pH 2.3.
Subject(s)
Flavin-Adenine Dinucleotide/chemistry , Hydrogen-Ion Concentration , Magnetic Fields , Quantum Theory , Water/chemistryABSTRACT
The photochemical reactions of blue-light receptor proteins have received much attention due to their very important biological functions. In addition, there is also growing evidence that the one particular class of such proteins, the cryptochromes, may be associated with not only a biological photo-response but also a magneto-response, which may be responsible for the mechanism by which many animals can respond to the weak geomagnetic field. Therefore, there is an important scientific question over whether it is possible to directly observe such photochemical processes, and indeed the effects of weak magnetic fields thereon, taking place both in purified protein samples in vitro and in actual biochemical cells and tissues. For the former samples, the key lies in being able to make sensitive spectroscopic measurements on very small volumes of samples at potentially low protein concentrations, while the latter requires, in addition, spatially resolved measurements on length scales smaller than typical cellular components, i.e., sub-micron resolution. In this work, we discuss a two- and three-color confocal pump-probe microscopic approach to this question which satisfies these requirements and is thus useful for experimental measurements in both cases.
Subject(s)
Absorption, Physicochemical , Flavins/chemistry , Magnetic Fields , Microscopy, Confocal/methods , Photochemical Processes , Color , Optical PhenomenaABSTRACT
Short-lived radicals generated in the photoexcitation of flavin adenine dinucleotide (FAD) in aqueous solution at low pH are detected with high sensitivity and spatial resolution using a newly developed transient optical absorption detection (TOAD) imaging microscope. Radicals can be studied under both flash photolysis and continuous irradiation conditions, providing a means of directly probing potential biological magnetoreception within sub-cellular structures. Direct spatial imaging of magnetic field effects (MFEs) by magnetic intensity modulation (MIM) imaging is demonstrated along with transfer and inversion of the magnetic field sensitivity of the flavin semiquinone radical concentration to that of the ground state of the flavin under strongly pumped reaction cycling conditions. A low field effect (LFE) on the flavin semiquinone-adenine radical pair is resolved for the first time, with important implications for biological magnetoreception through the radical pair mechanism.
