The Tryptophan Fluorescence of Tetracarbidium conophorum Agglutinin II and a Solution-Based Assay for the Binding of a Biantennary Glycopeptide

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299–303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (λ_ex = 295 nm, λ_em = 350 nm) decay is complex and can be described by four decay times with the following values: τ₁ = 7.4nsec, α₁ = 0.22; τ₂ = 2.9 nsec, α₂ = 0.25; τ₃ = l.0 nsec, α₃ = 0.34; τ₄ = 0.2 nsec, α₄ = 0.18. The addition of a biantennary glycopeptide $\begin{array}{*{20}c} {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 6)\neg } \\ {Man\beta (1 \to 4)GlcNAC\beta (1 \to 4)GlcAc\beta (1 \to )\begin{array}{*{20}c} {Glu - Nh_2 } \\ | \\ {Asn} \\ | \\ {COOH} \\ \end{array} } \\ {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 3)} \\ \end{array} $ to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8×10⁵ M^?1. The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.     

The Tryptophan Fluorescence of Tetracarbidium conophorum Agglutinin II and a Solution-Based Assay for the Binding of a Biantennary Glycopeptide

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299–303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (_ex = 295 nm, _em = 350 nm) decay is complex and can be described by four decay times with the following values: ₁ = 7.4nsec, ₁ = 0.22; ₂ = 2.9 nsec, ₂ = 0.25; ₃ = l.0 nsec, ₃ = 0.34; ₄ = 0.2 nsec, ₄ = 0.18. The addition of a biantennary glycopeptide to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8×10⁵ M^–1. The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.     

Mechanism of the efficient tryptophan fluorescence quenching in human gammaD-crystallin studied by time-resolved fluorescence

Human gammaD-crystallin (HgammaD-Crys) is a two-domain, beta-sheet eye lens protein found in the lens nucleus. Its long-term solubility and stability are important to maintain lens transparency throughout life. HgammaD-Crys has four highly conserved buried tryptophans (Trps), with two in each of the homologous beta-sheet domains. In situ, these Trps will be absorbing ambient UV radiation that reaches the lens. The dispersal of the excited-state energy to avoid covalent damage is likely to be physiologically relevant for the lens crystallins. Trp fluorescence is efficiently quenched in native HgammaD-Crys. Previous steady-state fluorescence measurements provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain [Chen, J., et al. (2006) Biochemistry 45, 11552-11563]. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations indicated that the fluorescence of Trp68 and Trp156 is quenched by fast electron transfer to the amide backbone. Here we report additional information obtained using time-resolved fluorescence spectroscopy. In the single-Trp-containing proteins (Trp42-only, Trp68-only, Trp130-only, and Trp156-only), the highly quenched Trp68 and Trp156 have very short lifetimes, tau approximately 0.1 ns, whereas the moderately fluorescent Trp42 and Trp130 have longer lifetimes, tau approximately 3 ns. In the presence of the energy acceptor (Trp68 or Trp156), the lifetime of the energy donor (Trp42 or Trp130) decreased from approximately 3 to approximately 1 ns. The intradomain energy transfer efficiency is 56% in the N-terminal domain and is 71% in the C-terminal domain. The experimental values of energy transfer efficiency are in good agreement with those calculated theoretically. The absence of a time-dependent red shift in the time-resolved emission spectra of Trp130 proves that its local environment is very rigid. Time-resolved fluorescence anisotropy measurements with the single-Trp-containing proteins, Trp42-only and Trp130-only, indicate that the protein rotates as a rigid body and no segmental motion is detected. A combination of energy transfer with electron transfer results in short excited-state lifetimes of all Trps, which, together with the high rigidity of the protein matrix around Trps, could protect HgammaD-Crys from excited-state reactions causing permanent covalent damage.     

Characterization of fluorescence quenching in bifluorophoric protease substrates

NorFES is a relatively rigid, bent undecapeptide which contains an amino acid sequence that is recognized by the serine protease elastase (AspAlaIleProNle downward arrow SerIleProLysGlyTyr ( downward arrow indicates the primary cleavage site)). Covalent attachment of a fluorophore on each side of NorFES's elastase cleavage site enables one to use a change of fluorescence intensity as a measure of enzymatic activity. In this study two bichromophoric NorFES derivatives, D-NorFES-A and D-NorFES-D, were prepared in which D (donor) was tetramethylrhodamine and A (acceptor) was rhodamine-X, two chromophores with characteristics suitable for energy transfer. Absorption and fluorescence spectra were obtained with both the intact and cleaved homodoubly, heterodoubly and singly labeled derivatives. It was found that both the homo and hetero doubly-labeled derivatives form ground-state complexes which exhibit exciton bands. The hetero labeled derivative exhibits little or no resonance energy transfer. Spectral measurements were also done in urea, which partially disrupts ground-state dimers.     

The identification of tryptophan residues responsible for ATP-induced increase in intrinsic fluorescence of myosin subfragment 1

ATP binding to myosin subfragment 1 (S1) induces an increase in tryptophan fluorescence. Chymotryptic rabbit skeletal S1 has 5 tryptophan residues (Trp113, 131, 440, 510 and 595), and therefore the identification of tryptophan residues perturbed by ATP is quite complex. To solve this problem we resolved the complex fluorescence spectra into log-normal and decay-associated components, and carried out the structural analysis of the microenvironment of each tryptophan in S1. The decomposition of fluorescence spectra of S1 and S1-ATP complex revealed 3 components with maxima at ca. 318, 331 and 339-342 nm. The comparison of structural parameters of microenvironment of 5 tryptophan residues with the same parameters of single-tryptophan-containing proteins with well identified fluorescence properties applying statistical method of cluster analysis, enabled us to assign Trp595 to 318 nm, Trp440 to 331 nm, and Trp 13, 131 and 510 to 342 nm spectral components. ATP induced an almost equal increase in the intensities of the intermediate (331 nm) and long-wavelength (342 nm) components, and a small decrease in the short component (318 nm). The increase in the intermediate component fluorescence most likely results from an immobilization of some quenching groups (Met437, Met441 and/or Arg444) in the environment of Trp440. The increase in the intensity and a blue shift of the long component might be associated with conformational changes in the vicinity of Trp510. However, these conclusions can not be extended directly to the other types of myosins due to the diversity in the tryptophan content and their microenvironments.     

Fluorescence analysis of a dynamic loop in the PCAF/GCN5 histone acetyltransferase

PCAF and GCN5 are histone acetyltransferase (HAT) paralogs which play roles in the remodeling of chromatin in health and disease. Previously, a conformationally flexible loop in the catalytic domain had been observed in the X-ray structures of GCN5 in different liganded states. Here, the conformation and dynamics of this PCAF/GCN5 alpha5-beta6 loop was investigated in solution using tryptophan fluorescence. A mutant human PCAF HAT domain (PCAF(Wloop)) was created in which the natural tryptophan (Trp-514) remote from the alpha5-beta6 loop was replaced with tyrosine and a glutamate within the loop (Glu-641) was substituted with tryptophan. This PCAF(Wloop) protein exhibited catalytic parameters within 3-fold of those of the wild-type PCAF catalytic domain, suggesting that the loop mutation was not deleterious for HAT activity. While saturating CoASH induced a 30% quenching of Trp fluorescence in PCAF(Wloop), binding of the high-affinity bisubstrate analogue H3-CoA-20 led to a 2-fold fluorescence increase. These different effects correlate with the different alpha5-beta6 loop conformations seen previously in X-ray structures. On the basis of stopped-flow fluorescence studies, binding of H3-CoA-20 to PCAF(Wloop) proceeds via a rapid association step followed by a slower conformational change involving loop movement. Time-resolved fluorescence measurements support a model in which the alpha5-beta6 loop in the H3-CoA-20-PCAF(Wloop) complex exists in a narrower ensemble of conformations compared to free PCAF(Wloop). The relevance of loop dynamics to PCAF/GCN5 catalysis and substrate specificity are discussed.     

Protease activity of Klebsiella pneumoniae of different virulence

The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.     

R150A mutant of F TraI relaxase domain: reduced affinity and specificity for single-stranded DNA and altered fluorescence anisotropy of a bound labeled oligonucleotide

F factor TraI is a helicase and a single-stranded DNA nuclease ("relaxase") essential for conjugative DNA transfer. A TraI domain containing relaxase activity, TraI36, was generated previously. Substituting Ala for Arg150 (R150A) of TraI36 reduces in vitro relaxase activity. The mutant has reduced affinity, relative to wild type, for a 3'-TAMRA-labeled 22-base single-stranded oligonucleotide. While both R150A and wild-type TraI36 bind oligonucleotide, only wild type increases steady-state fluorescence anisotropy of the labeled 22-base oligonucleotide upon binding. In contrast, binding by either protein increases steady-state anisotropy of a 3'-TAMRA-labeled 17-base oligonucleotide. Time-resolved intensity data for both oligonucleotides, bound and unbound, require three lifetimes for adequate fits, at least one more than the fluorophore alone. The preexponential amplitude for the longest lifetime increases upon binding. Time-resolved anisotropy data for both oligonucleotides, bound and unbound, require two rotational correlation times for adequate fits. The longer correlation time increases upon protein binding. Correlation times for the protein-bound 17-base oligonucleotide are similar for both proteins, with the longer correlation time in the range of molecular tumbling of the protein-DNA complex. In contrast, protein binding causes less dramatic increases in correlation times for the 22-base oligonucleotide relative to the 17-base oligonucleotide. Binding studies indicate that R150 contributes to recognition of bases immediately 3' to the DNA cleavage site, consistent with the apparent proximity of R150 and the 3' oligonucleotide end. Models in which the R150A substitution alters single-stranded DNA flexibility at the oligonucleotide 3' end or affects fluorophore-DNA or fluorophore-protein interactions are discussed.     

Cytotoxic properties of a recombinant hybrid of the A protein of Staphylococcus aureus with a fragment of exotoxin A of Pseudomonas aeruginosa]

By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and .Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.     

Pan1 is an intrinsically disordered protein with homotypic interactions

The yeast scaffold protein Pan1 contains two EH domains at its N‐terminus, a predicted coiled‐coil central region, and a C‐terminal proline‐rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp³¹² and Trp⁶⁴² are each in an EH domain, Trp⁹⁵⁷ is in the central region, and Trp¹²⁸⁰ is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern‐Volmer constants due to unique electrostatic environments of the tryptophan residues. Time‐resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis. Proteins 2013; 81:1944–1963. © 2013 Wiley Periodicals, Inc.