Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295nm light-emitting diode and time-correlated single-photon counting

McGuinness, Colin D.; Sagoo, Kulwinder; McLoskey, David; Birch, David J. S.

doi:10.1063/1.1984088

Cited by 53 publications

(28 citation statements)

References 15 publications

(12 reference statements)

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“…To address this, we conducted time-resolved fluorescence spectroscopic experiments at room temperature in solution with both WT and W285F 24 . Upon excitation by sub-nanosecond pulsed UV light 25 , we recorded time-courses of fluorescence decay, from which the time constants associated with distinct decay species were determined.…”

Section: Resultsmentioning

confidence: 99%

Dynamic crystallography reveals early signalling events in ultraviolet photoreceptor UVR8

Zeng

Ren²,

et al. 2015

Nature Plants

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Arabidopsis thaliana UVR8 (AtUVR8) is a long-sought-after photoreceptor that undergoes dimer dissociation in response to UV-B light. Crystallographic and mutational studies have identified two crucial tryptophan residues for UV-B responses in AtUVR8. However, the mechanism of UV-B perception and structural events leading up to dimer dissociation remain elusive at the molecular level. We applied dynamic crystallography to capture light-induced structural events in photoactive AtUVR8 crystals. Here we report two intermediate structures at 1.67Å resolution. At the epicenter of UV-B signaling, concerted motions associated with Trp285/Trp233 lead to ejection of a water molecule, which weakens an intricate network of hydrogen bonds and salt bridges at the dimer interface. Partial opening of the β-propeller structure due to thermal relaxation of conformational strains originating in the epicenter further disrupts the dimer interface and leads to dimer dissociation. These dynamic crystallographic observations provide structural insights into the photo-perception and signaling mechanism of UVR8.

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Section: Resultsmentioning

confidence: 99%

Dynamic crystallography reveals early signalling events in ultraviolet photoreceptor UVR8

Zeng

Ren²,

et al. 2015

Nature Plants

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“…Fluorescence decay measurements were performed using time-correlated single-photon counting [17] with a Horiba Jobin Yvon IBH FluoroCube. Excitation was with either a 1 MHz NanoLED [18] laser diode emitting at 452 nm and < 200 ps fwhm or a 40 MHz DeltaDiode at 378 nm.…”

Section: Methodsmentioning

confidence: 99%

Metal ion influence on eumelanin fluorescence and structure

Sutter¹,

Birch²

2014

Methods Appl. Fluoresc.

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Melanin has long been thought to have an unworkably weak and complex fluorescence, but here we study its intrinsic fluorescence in order to demonstrate how metal ions can be used to control the rate of formation, constituents and structure of eumelanin formed from the well-known laboratory auto-oxidation of 3, 4-dihydroxy-L-phenylalanine (L-DOPA). The effect on eumelanin absorption and fluorescence of a range of solvated metal ions is reported including Cu, Zn, Ni, Na and K. Monovalent cations and Zn have little effect, but the effect of transition metal cations can be considerable. For example, at pH 10, copper ions are shown to accelerate the onset of eumelanin formation, but not the rate of formation once it commences, and simplify the usual complex structure and intrinsic fluorescence of eumelanin in a way that is consistent with an increased abundance of 5,5-dihydroxyindole-2-carboxylic acid (DHICA). The presence of a dominant 6 ns fluorescence decay time at 480 nm, when excited at 450 nm describes a distinct photophysically species, which we tentatively assign to small oligomers. Copper is well-known to normally quench fluorescence, but increasing amounts of copper surprisingly leads to an increase in the fluorescence decay time of eumelanin, while reducing the fluorescence intensity, suggesting copper modification of the excited state. Such results have bearing on diverse areas. The most accepted morphology for melanin is that of a graphite-like sheet structure, and one which readily binds metal ions, an interaction that is thought to have an important, though as yet unclear bearing on several areas of medicine including neurology. There is also increasing interest in bio-mimicry by preparing and labelling sheet structures with metal ions for new electronic and photonic materials.

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“…[35] The excitation light was wavelength selected using a monochromator of bandwidth 4 nm. Fluorescence was collected at "magic angle" (54.78) at wavelength > 325 nm with a WG325 cut-off filter (Schott, Germany) placed on the emission window.…”

Section: Methodsmentioning

confidence: 99%

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

et al. 2009

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Structure of immobilised enzymes: Circular dichroism, infrared and fluorescence spectroscopic methods are used to characterise in situ structural stability of enzymes immobilised on particles. The enzyme subtilisin Carlsberg exhibits a similar secondary structure in solution and in the immobilised state but on the surface of porous silica gel a rigid tertiary structure is preferred (see picture). We report the development of biophysical techniques based on circular dichroism (CD), diffuse reflectance infrared Fourier transform (DRIFT) and tryptophan (Trp) fluorescence to investigate in situ the structure of enzymes immobilised on solid particles. Their applicability is demonstrated using subtilisin Carlsberg (SC) immobilised on silica gel and Candida antartica lipase B immobilised on Lewatit VP.OC 1600 (Novozyme 435). SC shows nearly identical secondary structure in solution and in the immobilised state as evident from far UV CD spectra and amide I vibration bands. Increased near UV CD intensity and reduced Trp fluorescence suggest a more rigid tertiary structure on the silica surface. After immobilised SC is inactivated, these techniques reveal: a) almost complete loss of near UV CD signal, suggesting loss of tertiary structure; b) a shift in the amide I vibrational band from 1658 cm−1 to 1632 cm−1, indicating a shift from α‐helical structure to β‐sheet; c) a substantial blue shift and reduced dichroism in the far UV CD, supporting a shift to β‐sheet structure; d) strong increase in Trp fluorescence intensity, which reflects reduced intramolecular quenching with loss of tertiary structure; and e) major change in fluorescence lifetime distribution, confirming a substantial change in Trp environment. DRIFT measurements suggest that pressing KBr discs may perturb protein structure. With the enzyme on organic polymer it was possible to obtain near UV CD spectra free of interference by the carrier material. However, far UV CD, DRIFT and fluorescence measurements showed strong signals from the organic support. In conclusion, the spectroscopic methods described here provide structural information hitherto inaccessible, with their applicability limited by interference from, rather than the particulate nature of, the support material.

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Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295nm light-emitting diode and time-correlated single-photon counting

Cited by 53 publications

References 15 publications

Dynamic crystallography reveals early signalling events in ultraviolet photoreceptor UVR8

Dynamic crystallography reveals early signalling events in ultraviolet photoreceptor UVR8

Metal ion influence on eumelanin fluorescence and structure

Optical Spectroscopic Methods for Probing the Conformational Stability of Immobilised Enzymes

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