Search

After more than one-half century of investigations, the mechanism of bioluminescence from the FMNH 2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase-bound FMNH-OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types., (© 2018 The American Society of Photobiology.)

Flavodoxins have a protein topology that can be traced back to the universal ancestor of the three kingdoms of life. Proteins with this type of architecture tend to temporarily misfold during unassisted folding to their native state and form intermediates. Several of these intermediate species are molten globules (MGs), which are characterized by a substantial amount of secondary structure, yet without the tertiary side-chain packing of natively folded proteins. An off-pathway MG is formed at physiological ionic strength in the case of the F44Y variant of Azotobacter vinelandii apoflavodoxin (i.e., flavodoxin without flavin mononucleotide (FMN)). Here, we show that at this condition actually two folding species of this apoprotein co-exist at equilibrium. These species were detected by using a combination of FMN fluorescence quenching upon cofactor binding to the apoprotein and of polarized time-resolved tryptophan fluorescence spectroscopy. Besides the off-pathway MG, we observe the simultaneous presence of an on-pathway folding intermediate, which is native-like. Presence of concurrent intermediates at physiological ionic strength enables future exploration of how aspects of the cellular environment, like for example involvement of chaperones, affect these species.

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.

Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer poly(2-methyl-vinyl-pyridinium) 41 -b-poly(ethylene-oxide) 205 . This packaging resulted in deviating spectroscopic features of the encapsulated EGFP molecules. Here we show that for monomeric EGFP variant (mEGFP) micellar encapsulation affects the absorption and fluorescence properties to a much lesser extent, and that changes in circular dichroism characteristics are specific for encapsulated EGFP. Time-resolved fluorescence anisotropy of encapsulated (m)EGFP established the occurrence of homo-FRET (Förster resonance energy transfer) with larger transfer correlation times in the case of EGFP. Together, these findings support that EGFP dimerizes whereas the mEGFP mainly remains as a monomer in the densely packed C3Ms. We propose that dimerization of encapsulated EGFP causes a reorientation of Glu222, resulting in a pK a shift of the chromophore, which is fully reversible after release of EGFP from the C3Ms at a high ionic strength.

The intrinsic fluorescence of tryptophan is frequently used to investigate the structure of proteins. The analysis of tryptophan fluorescence data is challenging: fluorescence (anisotropy) decays typically have multiple lifetime (correlation time) components and fluorescence spectra are broad and exhibit only minor shifts. In this work, we show that phasor approaches can substantially simplify tryptophan fluorescence analysis. To demonstrate this, we re-analyse previously recorded datasets of the denaturant (guanidinium hydrochloride, GuHCl) induced unfolding of a single-tryptophan-containing variant of apoflavodoxin from Azotobacter vinelandii. For three methods-(1) time-resolved fluorescence, (2) time-resolved fluorescence anisotropy and (3) steady-state fluorescence spectroscopy-we show that the phasor analysis can readily identify the presence of a folding intermediate. Moreover, the fractional contributions of protein states at various stages of unfolding and the values of the free energy difference of the unfolding process [Formula: see text] are obtained. The outcomes are compared to the global analysis results published previously.

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37+/-3 A at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.

In this paper spectroscopic data of a proteolytic enzyme adsorbed on solid-liquid interfaces are discussed. The experiments consisted of time-resolved and steady-state fluorescence of tryptophan residues and of circular dichroism (CD) which give information on the tertiary and secondary state of the protein, respectively. The spectroscopic properties are measured for the inhibited form of subtilisin 309 in situ on a hydrophilic silica surface and on a hydrophobic Teflon surface. The results are compared with those obtained for the protein in solution. In the case of fluorescence it is reasoned that the average excited-state lifetime and short internal rotation correlation times are indicative parameters for structural changes in the protein. The internal rotation is superimposed on the rotation of the adsorbed protein which is immobile on the fluorescence time scale. Fluorescence and CD both prove that the protein alters its conformation when it adsorbs at low surface coverage on hydrophobic Teflon particles. In that case the tryptophan fluorescence lifetime is shortened which is accompanied by an increase in the alpha-helix content. At monolayer coverage the protein maintains its original structure, although minor changes in fluorophore dynamics occur. On hydrophilic silica particles the results from both techniques do not point in the same direction. The fluorescence was not affected, irrespective of the surface occupation, while the CD experiments show a decrease in alpha-helix content at low surface coverage. Copyright 1997 Academic Press.