Blue Light Using flavin (BLUf) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light-and darkadapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule. Flavins are found in more than 370 enzymes 1 but only a few of them are photoactive 2,3. Three major families of photoreceptors which utilize flavin as a cofactor and whose functions are triggered by absorption of light are the photolyase/cryptochromes, the light oxygen voltage (LOV) domains and the blue light sensors using flavin (BLUF) proteins. Their photochemistry, though is rather diverse. In photolyases and cryptochromes, FAD (flavin adenine dinucleotide) is reduced via electron transfer through a tryptophan triad 4-6. Photolyases use light to repair UV-damaged DNA 7 whereas the proposed functions of cryptochromes range from setting the circadian clock in insects to sensing the weak magnetic field of Earth in migrating birds 6. In the LOV domains, the flavin cofactor is excited to a triplet state upon blue light absorption, followed by formation of a signalling state, characterized by a covalent bond between the flavin and a nearby cysteine residue, leading to the enhancement of the phototropin kinase activity 3. In BLUF domains, blue light excitation results in a signalling state (light-adapted state) that is characterized by a reorganization of the hydrogen bond network around FAD and the Tyr-Gln-Trp (Met) tetrad (Fig. 1). This is revealed by a characteristic 10-15 nm red-shift of the first π → π* transition and a 20 cm −1 downshift of the flavin C4=O stretching vibration compared to the dark-adapted state 8,9. In AppA BLUF , site directed mutagenesis has shown that Y21 and Q63 play a crucial role during photoactivation as the red shift upon illumination disappears if one of these r...

Class II malocclusion is one of the most common dental anomalies and the use of intermaxillary elastomers is the standard method in its treatment. However, orthodontic elastics cannot exert continuous force over a period of time due to force degradation. Our goal was to mechanically characterize the different types of elastomers during static and cyclic loads, based on uniform methodology and examine the morphological changes after loading. Ten types of latex-containing and four latex-free intermaxillary elastics were examined from six different manufacturers. To determine the mechanical characteristics of the elastomers, tensile tests, cyclical tensile fatigue tests and 24 h relaxation tests were performed, and the elastics were also subjected to scanning electron microscopy (SEM) and Raman spectroscopy. Regardless of the manufacturer, the latex-containing elastomers did not show significant differences in the percentage of elongation at break during the tensile test. Only one type of latex-containing elastomer did not tear during the 24 h cyclical fatigue test. Fatigue was confirmed by electron microscopy images, and the pulling force reduced significantly. During the force relaxation test, only one latex-free ligature was torn; the force degradation was between 7.8% and 20.3% for latex ligatures and between 29.6% and 40.1% for latex-free elastomers. The results showed that dynamic loading was more damaging to ligatures than static loading, latex-containing elastomers were more resistant than latex-free elastics, and which observation could have clinical consequences or a potential effect on patient outcome.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

No abstract