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We have cloned a novel fluorescent protein from the jellyfish Rhacostoma atlantica. The closest known related fluorescent protein is the Phialidium yellow fluorescent protein, with only a 55% amino acid sequence identity. A somewhat unusual alanine-tyrosine-glycine amino acid sequence forms the presumed chromophore of the novel protein. The protein has an absorption peak at 466 nm and a fluorescence emission peak at 498 nm. The fluorescence quantum yield was measured to be 0.77 and the extinction coefficient is 58 200 M(-1) cm(-1) . Several mutations were identified that shift the absorption peak to about 494 nm and the emission peak to between 512 and 514 nm.

With a view to assessing the current boundaries of non-invasive sensing of metabolites we compare the photophysical properties of melanin nanoparticles in vivo, size selected in natural form from Sepia officinalis extracted from cuttlefish, and synthesised from L-dihydoxyphenylalanine (DOPA) in water pools of reverse micelles. The fluorescence emission spectra and excited state decay components correlate well with the particle size. Recent developments in optical technology are discussed in the context of the formidable challenges and opportunities for metabolic sensing presented by endogenous fluorophores.

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.

The use of molecular genetics for introducing fluorescent molecules enables the use of donor–donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor–donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the C α -atoms of the positions V106C–H185C, H185C–M266C, and M266C–V106C were 60.9, 30.8, and 55.1 Å, respectively. These are in good agreement with the distances of 54 ± 4, 38 ± 3, and 55 ± 3 Å, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the C α -atoms physically cannot coincide, there is a reasonable agreement between the methods.

Structural insights into serpin-protease complexes reveal the inhibitory mechanism of serpins.

Plasminogen activator inhibitor type 1 (PAI-1) is a fast acting inhibitor of plasminogen activators (PAs). In accordance with other serpins, PAI-1 is thought to undergo a conformational change upon reactive center cleavage. In this study we have developed methods to produce and purify reactive center cleaved wild-type PAI-1 and characterized this molecular form of PAI-1 by biochemical and biophysical methods. Incubation with Sepharose-bound trypsin caused cleavage only at the P1-P1' bond in the reactive center and resulted in 39- and 4-kDa polypeptides, strongly held together by noncovalent interactions. Circular dichroism measurements suggest that the reactive center cleavage triggers larger conformational changes than the conversion from the active to the latent form. Cleaved PAI-1 did not bind to either PAs or vitronectin but retained the heparin-binding capacity. To study the structure of cleaved PAI-1 by polarized fluorescence spectroscopy and to measure intramolecular distances, we used cysteine substitution mutants to which extrinsic fluorescence probes were attached. These studies revealed increasing orientational freedom of probes in the P3 and P1' positions upon cleavage. Distance measurements based on fluorescence energy transfer between probes in positions P3 and P1' indicate that these residues are separated by at least 68 +/- 10 A in cleaved PAI-1.

In a previous report by us [1], it was demonstrated that the metal enhanced fluorescence (MEF) signal from fluorophores close to metal nanoparticles increased non-linearly with the far-field excitation power (irradiance). The effect was interpreted as an increase in the near-field excitation volume surrounding the metallic nanoparticles and was subsequently referred to as an excitation volumetric effect (EVE), where the technique would potentially allow for the near-field tuning of fluorescence enhancement factors, when observed from the far-field.In this contribution we show that the EVE phenomenon also has a profound effect on the formation of Reactive Oxygen Species (ROS), such as singlet oxygen (1O2) and superoxide anion radical (O2-∗), when sensitizers are placed in close proximity to plasmon supporting substrates. In particular, when the singlet oxygen sensitizer Rose Bengal is placed on a SiFs surface, i.e. on a silver island film [2], the 1O2 response to power is non-linear, and at 100 mW excitation power (535 nm) it is about 5 times higher, as compared to glass reference samples, measured with the commercially available 1O2 probe Sensor Green (SG). We also report a similar power dependence of superoxide generation for Acridine on SiFs surfaces, but using the dihydroethidium O2-∗probe (DHE). We additionally report the influence of various solvents where the singlet oxygen lifetime varies, i.e., water and deuterium oxide.[1] A. I. Dragan and C. D. Geddes, “Excitation volumetric effects (EVE) in metal-enhanced fluorescence,” Phys. Chem. Chem. Phys., vol. 13, no. 9, pp. 3831-3838, 2011.[2] SiFs paper reference here With rod.

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

In recent years our laboratory and others have demonstrated many examples and concepts in Metal-Enhanced Fluorescence1 (MEF), a surface plasmon phenomenon, which amplifies both fluorescence and luminescence signatures in the near-field, i.e. less than one wavelength of light away from a metallic object1. In all of these examples of MEF, and for over a decade, the fluorescence spectra has simply been reported as being enhanced, i.e. the emission is greater from a plasmonic substrate as compared to a suitable control sample.However, in this paper we will show that Metal-Enhanced Fluorescence from both a variety of plasmonic substrates and using a range of different fluorophores, often results in fluorophore spectral distortion2. More often than not, the red edge of the fluorescence spectra is observed to be distorted, as compared to the emission spectra of a fluorophore observed in the far-field and distal from plasmonic interactions. In addition, a significant MEF effect often results in notable changes in the spectrum full width at half maximum (FWHM). We discuss these new effects in terms of the mechanism of plasmonic enhancement.1Metal-Enhanced Fluorescence, Edited by Geddes, C.D., John Wiley and Sons, New Jersey, June 2010, 625 pgs, ISBN: 978-0-470-22838-8.2 Spectral Shifts in Metal-Enhanced Fluorescence, Karolin, J. and Geddes, C.D., (2014), Applied Physics Letters, 105, 063102.

We review the different approaches available to materials science using time-resolved fluorescence depolarization. Performance, limitations and challenges in this emerging area of nanometrology are discussed using the exemplars of sol–gel nanoparticle growth and protein dynamics.

In past several years, there has been significant investigation into the various synthetic routes of carbon nanodots along with their associated photophysical properties [1-3]. Carbon nanodots are naturally fluorescing nanometer-sized particles with interesting and unique photophysical properties, which make them highly applicable for various applications in the life sciences [2-3]. Our lab has been investigating these particles produced by various combustion routes for many years, studying both the photophysical and plasmon-enhanced photophysical properties [1]. In order to fully understand the photophysical properties of carbon nanodots, in this poster we have examined the both the internal and external structure of these particles in an attempt to ascertain the origins of the fluorescence signature/s, using a combination of differently charged ions; which ultimately results in both static and dynamic quenching processes being observed. Our results reveal significant vibronic structure of the nanodots’ chromophore, which can readily be quenched by non-charged ions (acrylamide), suggesting a buried fluorescent chromophore center.[1] Y. Zhang, H. Goncalves, J. C. G. Esteves Da Silva, and C. D. Geddes, “Metal-enhanced photoluminescence from carbon nanodots,” Chem. Commun. 47, 5313-5315 (2011).[2] S. Baker and G. Baker, “Luminescent Carbon Nanodots: Emergent Nanolights,” Angew, Chem. Int. Ed. 49, 6726-6744 (2010).[3] H. Li, Z. Kang, Y. Liu, and S-T. Lee, “Carbon nanodots synthesis, properties and applications,” J. Mater. Chem, 22, 24230-24253 (2010).

Melanins are common pigments with a non-repeating primary structure that is generally accepted to be composed of dihydroxyindoles. However, despite intensive research the secondary structure defining the minimum functional unit (protomolecule) remains elusive. We have revisited eumelanin formation in-situ during the non-enzymatic auto-oxidation of 3,4-dihydroxy-L-phenylalanine by using the fluorescence of thioflavin T; an extrinsic probe known to report on sheet structure. This approach obviates the complex intrinsic fluorescence and reveals a sigmoidal temporal dependence of assembly that is consistent with protomolecule formation and assembly into a stacked sheet structure rather than a randomized heteropolymer formed by monomer addition.

A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Förster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.

Carbon nanodots, i.e. c-dots, have recently emerged as new class of fluorescent labels with tunable emission characteristics, high quantum yields and photostability, and with a low toxicity as compared to semiconductor quantum dots. In a previous report by Geddes and co-workers [1] it was demonstrated that the emission properties of c-dots can be further amplified by plasmon supporting materials, such as silver island films, however, it was reported that inhomogeneity due to the c-dot size distribution complicated the interpretation of the emission data. In the present report we apply the technique of fluorescence correlation spectroscopy, FCS, to characterize c-dots synthetically realized by the flame combustion of hydrocarbon/oxygen mixtures where particles are collected from the exhaust gases in liquid traps. We report a correlation between the c-dots particle size distribution with the stoichiometric composition of the flame and compare results with fluorescence anisotropy measurements.[1] Yongxia Zhang, Helena Goncalves, Joaquim C. G. Esteves da Silva and Chris D. Geddes, Metal-enhanced photoluminescence from carbon nanodots, Chem. Commun. , 2011, 47, 5313-5315.