Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of β-sheets into α-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p

Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We studied Hansenula polymorpha Pex5p (HpPex5p) using fluorescence spectroscopy. The intensity of Trp fluorescence of purified HpPex5p increased by 25% upon shifting the pH from pH 6.0 to pH 7.2. Together with the results of fluorescence quenching by acrylamide, these data suggest that the conformation of HpPex5p differs at these two pH values. Fluorescence anisotropy decay measurements revealed that the pH affected the oligomeric state of HpPex5p, possibly from monomers/dimers at pH 6.0 to larger oligomeric forms at pH 7.2. Addition of dansylated peptides containing a PTS1, caused some shortening of the average fluorescence lifetime of the Trp residues, which was most pronounced at pH 7.2. Our data are discussed in relation to a molecular model of HpPex5p based on the three-dimensional structure of human Pex5p

Spectral Characterization of Dictyostelium Autofluorescence

Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from ~500 to 650 nm with a maximum at ~510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5–7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, prebleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium.

Simultaneous analyses of fluorescence decay and anisotropy decay in green fluorescent protein dimer from jellyfish Clytia gregaria : FRET and molecular dynamics simulation

Structural and dynamic behaviors of the green fluorescent protein dimer from jellyfish Clytia gregaria (cgGFP) were investigated by means of molecular dynamics (MD) simulation. Both neutral and ionic forms of the chromophore, p-hydroxybenzylideneimidazolinone (GYS) were considered. The partial atomic charges of the chromophore were derived by BCC and RESP approaches. The structures were compared between the anionic and neutral cgGFP, and between the two subunits (Sub A and Sub B) of the protein dimer. The observed fluorescence intensity and anisotropy decays were further analyzed with theoretical expressions by employing the atomic coordinates of neutral cgGFP obtained by MD simulation. It was assumed that the fluorescence quenching of GYSA and GYSB is ascribed to HB formations between heteroatoms of GYSs and nearby amino acids. Excellent agreement between the observed and calculated intensity decays, and the observed and calculated anisotropy decays were obtained with RESP1 model. The agreements were better in RESP model than those in BCC one. Mean quenching constants of GYSA and GYSB were 0.27 and 0.59 ns−1 overall MD snapshots with RESP1. Mean value of square of direction cosine between the two transition moments of GYSs was 0.74, and that of square of orientation factor was 0.53, and the FRET rates from GYSA to GYSB, and from GYSB to GYSA were 0.87 and 1.87 ns−1

Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions

Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.

Dynamic Fluorescence Spectroscopy on Single Tryptophan Mutants of EIImtl in Detergent Micelles. Effects of Substrate Binding and Phosphorylation on the Fluorescence and Anisotropy Decay

The effects of substrate and substrate analogue binding and phosphorylation on the conformational dynamics of the mannitol permease of Escherichia coli were investigated, using time-resolved fluorescence spectroscopy on mutants containing five single tryptophans situated in the membrane-embedded C domain of the enzyme. Since no fluorescent impurities are present in these mutants, the changes in fluorescence and anisotropy could be related with changes in the tryptophan microenvironment. Tryptophans at positions 30 and 42 showed changes in fluorescence intensity decay upon binding mannitol, which were reflected in the changes in lifetime distribution patterns. The disappearance of the shortest-lived decay component in these mutants, as well as in the mutant with a single tryptophan at position 109, indicates a change in the local environment such that quenching via neighboring side chains or solvent is reduced. Phosphorylation at histidine 554 and cysteine 384, located in the cytoplasmatic A and B domains of EIImtl, respectively, induced an increase in the average fluorescence lifetimes of all of the tryptophans. The effect was most pronounced for tryptophans 30 and 109 which show large increases in the average fluorescence lifetime mainly due to loss of short-lived decay components. A correlation time distribution of the individual tryptophans deduced from an analysis of the anisotropy decay showed that they differed in their rotational mobility with tryptophan 30 showing the least local flexibility. Phosphorylation resulted in immobilization of W109 which, together with changes in the average fluorescence lifetime, is evidence for a conformational coupling between the phosphorylated B domain and the C domain. The influence of mannitol binding on the rotational behavior of the tryptophans is limited; it induces more internal flexibility at all tryptophan positions. A rotational correlation time of 30 ns was resolved for tryptophan 30, which probably represents a rotational mode of the micelle-embedded C-domain of EIImtl or a portion thereof. Upon phosphorylation, this rotational correlation time increases to 50 ns, probably reflecting a changed spatial orientation of W30 with respect to the C domain. Although kinetic experiments have shown that none of the tryptophans is essential for the catalytic activity of EIImtl, it is significant that the residues most sensitive to mannitol binding, W30 and W42, are both located in the first membrane-spanning α-helix, a portion of which is highly conserved among mannitol-specific EII’s of different bacteria.

The Mechanism of Bacterial Bioluminescence

Bioluminescence is a chemiluminescence in which one of the participants is a protein and where the light emission serves a functional purpose for the organism in which the protein is found. The nature of the protein depends on the organism. In the firefly, it is called firefly “luciferase” from the Latin, Lucifer: The Bringer of Light. Luciferase is a generic name, and the type of organism needs to precede the name because the bioluminescence property is usually specific for that type. The enzyme suffix “-ase” is often used not because turnover has been proven, in most cases a difficult task, but because of saturation kinetics observed for the light reaction