Combination Of Fluorescence Techniques Research Articles

Conformational Fluctuations as an Intrinsic Mechanism of Action: The Lipase-Specific Foldase of Pseudomonas Aeruginosa Studied by Hybrid Fluorescence Spectroscopy and MD Simulations

The description of protein function requires structural information, not only from static structural models but also of flexibility and dynamics. An investigation of highly flexible proteins or IDPs requires techniques sensitive to a variety of time- and distance-scales. Fluorescence (FRET, FCS) is known to provide good temporal and spatial coverage but it is a label-based technique and lacks atomistic-details. All-atom MD, on the other hand, is challenged by simulation of slow processes and accurate description of all interactions. The lipase-specific foldase (Lif) from Pseudomonas aeruginosa is a highly flexible protein with residual secondary and tertiary structure. It is mandatory to produce lipase A (LipA) in an enzymatically active conformation. Using a combination of fluorescence techniques (single molecule FRET, fluorescence lifetime analysis and fFCS) and all-atom MD we propose a description of structure and dynamics of Lif in relation to LipA. In the Lif:LipA complex, Lif forms flexible α-helical scaffold embracing LipA in headphone-like structure. In the unbound form, Lif does not stay in the hollow “headphone” conformation but rather exhibits large-scale conformational dynamics, where its α-helical structure undergoes reversible collapses and extensions as well as unfolding, on the sub-microsecond to sub-millisecond timescale. This process allows Lif to bind LipA despite the fickleness of structure. We show how the multitude of states in fast exchange provides a basis for binding. In addition we show how this hybrid approach benefits FRET (insight into the molecular structure) and MD (sampling).

Polymer-Coated Nanoparticles by Adsorption of Hydrophobically Modified Poly(N,N-dimethylacrylamide)

We prepared a reactive random copolymer of N-acryloxysuccinimide and N,N-dimethylacrylamide (DMA) by reversible addition-fragmentation chain transfer polymerization, with Mn ≈ 50k and 23 mol % reactive NAS groups. This copolymer was subsequently modified with hydrophobic (dodecyl) and fluorescent (pyrene, PY; phenanthrene, PHE; or anthracene, AN) side groups, to obtain fluorescent amphiphilic polymers with the same backbone and different substituents. These polymers were adsorbed onto model (ca. 130 nm diameter) poly(butyl methacrylate) nanoparticles, and the size and structure of the adsorbed layer were evaluated using a combination of fluorescence techniques and light scattering. The total diameter increases very fast with polymer concentration up to ca. 140 nm, and then more slowly to 154 nm, stabilizing at this value which corresponds to a polymer shell thickness of ca. 12 nm. In order to evaluate the distribution of hydrophobic groups on the adsorbed polymer layer, we used Förster resonance energy transfer between PHE- and AN-labeled poly(DMA) chains. The obtained concentration profile of the adsorbed polymer corresponds to a coated particle radius which is only slightly smaller than the hydrodynamic radius measured in the same conditions, indicating that the dyes are not located at the particle interface but mostly distributed across the adsorbed layer. Finally, we observed that hydrophobically modified PHE-labeled poly(DMA) chains adsorbed to the nanoparticles were very efficiently displaced by identical hydrophobically modified chains with five times their molecular weight (Mn ≈ 250k) but labeled with PY.

Fluorone dyes have binding sites on both cytoplasmic and extracellular domains of Na,K-ATPase

Combination of fluorescence techniques and molecular docking was used to monitor interaction of Na,K-ATPase and its large cytoplasmic loop connecting fourth and fifth transmembrane helices (C45) with fluorone dyes (i.e. eosin Y, 5(6)-carboxyeosin, rose bengal, fluorescein, and erythrosine B). Our data suggested that there are at least two binding sites for all used fluorone dyes, except of 5(6)-carboxyeosin. The first binding site is located on C45 loop, and it is sensitive to the presence of nucleotide. The other site is located on the extracellular part of the enzyme, and it is sensitive to the presence of Na+ or K+ ions. The molecular docking revealed that in the open conformation of C45 loop (which is obtained in the presence of ATP) all used fluorone dyes occupy position directly inside the ATP-binding pocket, while in the closed conformation (i.e. in the absence of any ligand) they are located only near the ATP-binding site depending on their different sizes. On the extracellular part of the protein, the molecular docking predicts two possible binding sites with similar binding energy near Asp897(α) or Gln69(β). The former was identified as a part of interaction site between α- and β-subunits, the latter is in contact with conserved FXYD sequence of the γ-subunit. Our findings provide structural explanation for numerous older studies, which were performed with fluorone dyes before the high-resolution structures were known. Further, fluorone dyes seem to be good probes for monitoring of intersubunit interactions influenced by Na+ and K+ binding.

Simultaneous spectrofluorimetric determination of levodopa and propranolol in urine using feed-forward neural networks assisted by principal component analysis

The simultaneous determination of levodopa (LD) and propranolol (PRO) using fluorescence spectrometric technique is described. The method involves measuring the natural fluorescence of these drugs in the micellar media of sodium dodecyl sulfate (SDS) using principal component analysis-feed-forward neural networks (PC-FFNNs). Experimental conditions such as effect of pH and SDS concentration were optimized. Under the optimum conditions, the linear determination ranges of LD and PRO are 2.0 × 10 −8 to 1.0 × 10 −5 mol L −1 and 3.6 × 10 −9 to 1.8 × 10 −6 mol L −1, respectively. A set of synthetic binary mixtures of LD and PRO was prepared and their concentrations were predicted by the proposed method. Satisfactory results were obtained by the combination of fluorescence technique with chemometrics methods. The method was successfully applied to the determination of LD and PRO in tap water and in urine samples.

Binding of Lysozyme to Phospholipid Bilayers: Evidence for Protein Aggregation upon Membrane Association

Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho- rac-glycerol, 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho- rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5′-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated protein species is likely to be the mechanism responsible for the cytotoxicity of lysozyme.

Identifying Transmembrane States and Defining the Membrane Insertion Boundaries of Hydrophobic Helices in Membrane-inserted Diphtheria Toxin T Domain

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.