Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134–206 of SNAP-25 (synaptosome-associated protein of 25 kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. In this article, we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarizations, and lifetimes.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity

The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)–fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134–206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V_max and k_cat) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.     

Fluorescent probe studies on binding of glyceraldehyde-3-phosphate dehydrogenase to phosphatidylinositol liposomes. Further evidence for conformational changes

Glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle can be absorbed on charged lipid bilayers by electrostatic forces. Upon binding to phosphatidylinositol liposomes the enzyme modifies its conformational state as it is shown by resonance energy transfer experiments. In the presence of 2-mercaptoethanol o-phthaldialdehyde reacts with amino groups of the protein and the covalently bound fluorophore is an acceptor of excitation energy transferred from tryptophanyl residues of the protein. The observed decrease of energy transfer efficiency upon binding to phosphatidylinositol liposomes is compared with the influence of the urea on the fluorescence spectra of the labelled protein. Significance of conformational changes of the enzyme upon adsorption on liposomes in the regulating function of cell membranes is discussed.     

L-selectin transmembrane and cytoplasmic domains are monomeric in membranes

A recombinant protein termed CLS, which corresponds to the C-terminal portion of human L-selectin and contains its entire transmembrane and cytoplasmic domains (residues Ser473-Arg542), has been produced and its oligomeric state in detergents characterized. CLS migrates in the SDS polyacrylamide gel at a pace that is typically expected from a complex twice of its molecular weight. Additional studies revealed, however, that this is due to residues in the cytoplasmic domain, as mutations in this region or its deletion significantly increased the electrophoretic rate of CLS. Analytical ultracentrifugation and fluorescence resonance energy transfer studies indicated that CLS reconstituted in dodecylphosphocholine detergent micelles is monomeric. When the transmembrane domain of L-selectin is inserted into the inner membrane of Escherichia coli as a part of a chimeric protein in the TOXCAT assay, little oligomerization of the chimeric protein is observed. Overall, these results suggest that transmembrane and cytoplasmic domains of L-selectin lack the propensity to self-associate in membranes, in contrast to the previously documented dimerization of the transmembrane domain of closely related P-selectin. This study will provide constraints for future investigations on the interaction of L-selectin and its associating proteins.     

Lipid association-induced N- and C-terminal domain reorganization in human apolipoprotein E3

Apolipoprotein E (apoE) is a 299 amino acid, anti-atherogenic protein that plays a key role in regulating plasma lipoprotein metabolism. It is composed of an N-terminal (NT) domain (residues 1-191) that is responsible for binding to members of the low density lipoprotein receptor family and a C-terminal (CT) domain (residues 216-299) that anchors the protein to lipoprotein particles by virtue of its high-affinity lipid binding characteristics. Isoform-specific differences in the NT domain that modulate the lipoprotein binding preference elicited by the CT domain suggest the existence and importance of domain interactions in this protein. Employing steady state fluorescence quenching and resonance energy transfer techniques, spatial proximity relationships between the N- and C-terminal domains were investigated in recombinant human apoE3. ApoE3 containing a single Trp at position 264 and an N-iodoacetyl-N'-(5-sulfo-1-napthyl) ethylenediamine (AEDANS) moiety covalently attached to the lone Cys residue at position 112 was used (AEDANS-apoE3/W@264). Fluorescence quenching studies revealed a solvent-exposed location for Trp-264. In the lipid-free state, fluorescence resonance energy transfer (FRET) was noted between Trp-264 and AEDANS, with a calculated distance of 27 A between the two fluorophores. Control experiments established that FRET observed in this system is intramolecular. FRET was abolished upon proteolysis in the linker region connecting the NT and CT domains. Lowering the solution pH to 4 induced an increase in the efficiency of intramolecular energy transfer, with the two domains reorienting about 5 A closer to one another. Interdomain FRET was retained in the presence of 0.6-1.0 m guanidine hydrochloride but was lost at higher concentrations, a manifestation of unfolding of the domains and increased distance between the donor-acceptor pair. Interaction of AEDANS-apoE3/W@264 with lipid induced a loss of FRET, attributed to spatial repositioning of the domains by >80 A. The data provide biophysical evidence that, in addition to reported conformational changes in the four-helix bundle configuration induced by lipid association, lipid binding of apoE is accompanied by reorientation of the tertiary disposition of the NT and CT domains.     

Alterations in photosynthetic pigments and amino acid composition of D1 protein change energy distribution in photosystem II

The marine cyanobacterium Prochlorococcus marinus accumulates divinyl chlorophylls instead of monovinyl chlorophylls to harvest light energy. As well as this difference in its chromophore composition, some amino acid residues in its photosystem II D1 protein were different from the conserved amino acid residues in other photosynthetic organisms. We examined PSII complexes isolated from mutants of Synechocystis sp. PCC 6803, in which chromophore and D1 protein were altered (Hisashi Ito and Ayumi Tanaka, 2011) to clarify the effects of chromophores/D1 protein composition on the excitation energy distribution. We prepared the mutants accumulating divinyl chlorophyll (DV mutant). The amino acid residues of V205 and G282 in the D1 protein were substituted with M205 and C282 in the DV mutant to mimic Prochlorococcus D1 protein (DV-V205M/G282C mutant). Isolated PSII complexes were analyzed by time-resolved fluorescence spectroscopy. Energy transfer in CP47 was interrupted in PSII containing divinyl chlorophylls. The V205M/G282C mutation did not recover the energy transfer pathway in CP47, instead, the mutation allowed the excitation energy transfer from CP43 to CP47, which neighbors in the PSII dimer. Mutual orientation of the subcomplexes of PSII might be affected by the substitution. The changes of the energy transfer pathways would reduce energy transfer from antennae to the PSII reaction center, and allow Prochlorococcus to acquire light tolerance.     

Comparative fluorescence properties of bovine,goat, human and guinea pig α lactalbumin: Characterization of the environments of individual tryptophan residues in partially folded conformers

α Lactalbumin exists as a partially folded conformer (U form) at acid pH. A second partially folded conformer (H form) is formed above 60°. Comparison of the changes in tryptophan fluorescence which occur on forming U and H for the bovine, goat, human and guinea pig proteins, as well as analysis of fluorescence properties for the bovine protein and an N bromo succinimide derivative of this protein, have made it possible to determine which tryptophan residues give rise to such changes in fluorescence, and to draw a distinction between the molecular structure of the U and H forms of the protein. Trp 28 and 109 in the native state transfer their excitation energy to trp 63 whose fluorescence is quenched by a pair of vicinal disulfide bridges. This process persists in the U form of the protein, but is absent in the H conformer. Most of the change in fluorescence seen in the N ? U conversion is due to increase in yield of trp 28, while the changes in fluorescence occurring on formation of the H form are due to exposure of trp 63 and elimination of its quenching and/or excited state transfer from 28 to 109.     

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.     

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the F_oF₁-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.     

Human serum albumin–malathion complex study in the presence of silver nanoparticles at different sizes by multi spectroscopic techniques

The binding of malathion to human serum albumin (HSA) in the presence of silver nanoparticles (AgNPs) was investigated for the first time by multiple spectroscopic methods such as fluorescence quenching, fluorescence resonance energy transfer (FRET), circular dichroism, red-edge excitation shift (REES), synchronous fluorescence and three dimensional fluorescence spectroscopy under physiological conditions .The results indicated that binding of malathion to HSA induced fluorescence quenching through static mechanism. The number of binding sites was calculated by double logarithmic equation. Changes in the micro-environment of the fluorophore residues were also probed by synchronous fluorescence spectroscopy and REES. Changes of secondary structure of HSA in HSA–malathion complex was verified by circular dichroism approach in the presence of AgNPs that showed the electrostatic interaction changes in the protein structure. The binding average distance (r) between the donor (HSA) and the acceptor (malathion) was measured and found to be 1.63?nm according to the Forster’s theory of non-radiation energy transfer which was <7?nm confirmed the existence of static quenching in the presence of AgNPs. The conformational changes of HSA by three-dimensional fluorescence spectroscopy were studied. By comparing the resonance light scattering in the binary and ternary systems, we could estimate the effect of AgNPs on the precipitation of the malathion on the HSA. Generally we have discussed the toxicity reduction effect of malathion in food industrial by the results of spectroscopy techniques.     

Location of the stilbenedisulfonate binding site of the human erythrocyte anion-exchange system by resonance energy transfer.

The stilbenedisulfonate inhibitory site of the human erythrocyte anion-exchange system has been characterized by using serveral fluorescent stilbenedisulfonates. The covalent inhibitor 4-benzamido-4'-isothiocyanostilbene-2,2'-disulfonate (BIDS) reacts specifically with the band 3 protein of the plasma membrane when added to intact erythrocytes, and the reversible inhibitors 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) and 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS) show a fluorescence enhancement upon binding to the inhibitory site on erythrocyte ghosts. The fluorescence properties of all three bound probes indicate a rigid, hydrophobic site with nearby tryptophan residues. The Triton X-100 solublized and purified band 3 protein has similar affinities for DBDS, BADS, and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS) to those observed on intact erythrocytes and erythrocyte ghosts, showing that the anion binding site is not perturbed by the solubilization procedure. The distance between the stilbenedisulfonate binding site and a group of cysteine residues on the 40 000-dalton amino-terminal cytoplasmic domain of band 3 was measured by the fluorescence resonance energy transfer technique. Four different fluorescent sulfhydryl reagents were used as either energy transfer donors or energy transfer acceptors in combination with the stilbenedisulfonates (BIDS, DBDS, BADS, and DNDS). Efficiencies of transfer were measured by sensitized emisssion, donor quenching, and donor lifetime changes. Although these sites are approachable from opposite sides of the membrane by impermeant reagents, they are separated by only 34--42 A, indicating that the anion binding site is located in a protein cleft which extends some distance into the membrane.     

Simultaneous DNA binding, bending, and base flipping: evidence for a novel M.EcoRI methyltransferase-DNA complex

We measured the kinetics of DNA bending by M.EcoRI using DNA labeled at both 5'-ends and observed changes in fluorescence resonance energy transfer. Although known to bend its cognate DNA site, energy transfer is decreased upon enzyme binding. This unanticipated effect is shown to be robust because we observe the identical decrease with different dye pairs, when the dye pairs are placed on the respective 3'-ends, the effect is cofactor- and protein-dependent, and the effect is observed with duplexes ranging from 14 through 17 base pairs. The same labeled DNA shows the anticipated increased energy transfer with EcoRV endonuclease, which also bends this sequence, and no change in energy transfer with EcoRI endonuclease, which leaves this sequence unbent. We interpret these results as evidence for an increased end-to-end distance resulting from M.EcoRI binding, mediated by a mechanism novel for DNA methyltransferases, combining DNA bending and an overall expansion of the DNA duplex. The M.EcoRI protein sequence is poorly accommodated into well defined classes of DNA methyltransferases, both at the level of individual motifs and overall alignment. Interestingly, M.EcoRI has an intercalation motif observed in the FPG DNA glycosylase family of repair enzymes. Enzyme-dependent changes in anisotropy and fluorescence resonance energy transfer have similar rate constants, which are similar to the previously determined rate constant for base flipping; thus, the three processes are nearly coincidental. Similar fluorescence resonance energy transfer experiments following AdoMet-dependent catalysis show that the unbending transition determines the steady state product release kinetics.     

Sensitive detection of the redox state of copper proteins using fluorescence

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.     

What causes the depolarization of trypsin and trypsinogen fluorescence. Intramolecular mobility or non-radiative energy transfer?

Analysis of protein data bank information about the coordinates of definite atoms of protein macromolecules provides an opportunity to evaluate the efficiency of non-radiative resonance energy transfer within the model of fixed, strictly oriented oscillators. Such evaluations for trypsin and trypsinogen (and also for trypsin complex with a pancreatic inhibitor) show that the efficiency of energy transfer among each pair of tryptophan residues is negligibly small. It is also shown that a fairly effective energy transfer from tyrosine to tryptophan residues is possible. The conclusions have been made that the Tyr-Trp energy transfer is one of the factors determining the shape of the trypsin polarization spectrum, and that upon fluorescence excitation at the long-wavelength edge of the absorption spectrum, the depolarization of trypsin fluorescence in aqueous solution at ambient temperature - compared to model compounds (tryptophan, N-acetyltryptophan, glycyltryptophan, etc.), under the conditions of infinite viscosity - is due to the Brownian rotational motion of the macromolecules as a whole as well as the intramolecular mobility. The differences in the level and character of intramolecular mobility of trypsin and trypsinogen are discussed.     

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of F(o)F(1)-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.     

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

The Archaebacterium Thermoplasma acidophilum has a histone-like protein (HTa) abundantly associated with its deoxyribonucleic acid. Each native tetrameric complex of HTa contains 20 phenylalanine residues, 4 tyrosine residues, and no tryptophan. When the protein was excited by radiation at 252 nm, which is a wavelength absorbed predominantly by phenylalanine, the fluorescent emission was mostly from tyrosine. According to the excitation spectrum for this tyrosine fluorescence, the cause was energy transfer from phenylalanine, which occurred with about 50% efficiency. When the tyrosine residues were removed enzymatically, the excited-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.     

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.     

A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes

A lipid transfer protein, purified from bovine brain (23.7 kDa, 208 amino acids) and specific for glycolipids, has been used to develop a fluorescence resonance energy transfer assay (anthrylvinyl-labeled lipids; energy donors and perylenoyl-labeled lipids; energy acceptors) for monitoring the transfer of lipids between membranes. Small unilamellar vesicles composed of 1 mol% anthrylvinyl-galactosylceramide, 1.5 mol% perylenoyl-triglyceride, and 97.5% 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) served as donor membranes. Acceptor membranes were 100% POPC vesicles. Addition of glycolipid transfer protein to mixtures of donor and acceptor vesicles resulted in increasing emission intensity of anthrylvinyl-galactosylceramide and decreasing emission intensity of the nontransferable perylenoyl-triglyceride as a function of time. The behavior was consistent with anthrylvinyl-galactosylceramide being transferred from donor to acceptor vesicles. The anthrylvinyl and perylenoyl energy transfer pair offers advantages over frequently used energy transfer pairs such as NBD and rhodamine. The anthrylvinyl emission overlaps effectively the perylenoyl excitation spectrum and the fluorescence parameters of the anthrylvinyl fluorophore are nearly independent of the medium polarity. The nonpolar fluorophores are localized in the hydrophobic region of the bilayer thus producing minimal disturbance of the bilayer polar region. Our results indicate that this method is suitable for assay of lipid transfer proteins including mechanistic studies of transfer protein function.