A non-linear least-squares approach to the resolution of heterogeneous fluorescence from multitryptophan proteins

Protein heterogeneous fluorescence results from the different microenvironment of each emitting chromophore. The structural and dynamic information contained in this emission can be extracted to some extent by selective quenching experiments. In this work, graphical and numerical methods are described for the analysis of protein emission in terms of three separated contributions: a fluorescence fraction which is not accessible to the quencher and two additional fractions with different solvent exposure. ‘Static quenching’ deviations from Stern-Volmer behaviour are also discussed. The application of these methods is exemplified on simulated quenching experiments and real data on acrylamide quenching of lysozyme fluorescence.     

The sensitive determination of nucleic acids using a fluorescence-quenching method.

Experiments indicated that nucleic acids can quench the fluorescence of the Eu3+ -2-thenoyltrifluoroacetone (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids was proposed. The experiments indicated that under the optimum conditions, the quenched fluorescence intensity was in proportion to the concentration of nucleic acids in the range 1.0 x 10(-11)-1.0 x 10(-6) g/mL for yeast RNA (yRNA), 5.0 x 10(-11)-5.0 x 10(-7) g/mL for fish sperm (fsDNA) and 1.0 x 10(-10)-1.5 x 10(-6) g/mL for calf thymus DNA (ctDNA). Their detection limits were 3.0 x 10(-12), 4.0 x 10(-12) and 5.0 x 10(-11) g/mL, respectively. Therefore, the proposed method is one of the most sensitive methods available. The interaction between nucleic acids and Eu3+ -TTA-Phen is also discussed.     

Penetration of dioxygen into proteins studied by quenching of phosphorescence and fluorescence

Experiments were done to measure the ability of dioxygen to collisionally quench the phosphorescent and fluorescent tryptophans in alcohol dehydrogenase and alkaline phosphatase. In all cases, luminescence is quenched with rate constants close to 1 x 10(9) M-1 s-1. The rate of reaching the buried tryptophans is little affected by solvent viscosity due to added glycerol. Quenching by dioxygen is not due to a protein-opening reaction. It appears to be rate limited by internal protein diffusion rather than at the entry step. Dioxygen appears to enter the proteins directly, as in liquidlike diffusion, rather than through transiently forming channels that are only present a small fraction of the time. A high-pressure oxygen system is described that considerably facilitates fluorescence quenching experiments.     

Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)]=10:1. In the linear range (0-50 micromol L(-1)) quenching constants were calculated using Stern-Volmer equation. Temperature in the range 298 K-310 K had a significant effect (p<0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

Interaction of loratadine with serum albumins studied by fluorescence quenching method

The interactions between loratadine and bovine serum albumin (BSA) and human serum albumin (HSA) were studied using tryptophan fluorescence quenching method. The fluorescence intensity of the two serum albumins could be quenched 70% at the molar ratio [loratadine]:[BSA (or HSA)] = 10:1. In the linear range (0–50 μmol L^− 1) quenching constants were calculated using Stern–Volmer equation. Temperature in the range 298 K–310 K had a significant effect (p < 0.05) on the two serum albumins through ANOVA analysis and t-test. Furthermore the conformation changes in the interactions were studied using FTIR spectroscopy.     

DNA bending induced by high mobility group proteins studied by fluorescence resonance energy transfer.

The HMG domains of the chromosomal high mobility group proteins homologous to the vertebrate HMG1 and HMG2 proteins preferentially recognize distorted DNA structures. DNA binding also induces a substantial bend. Using fluorescence resonance energy transfer (FRET), we have determined the changes in the end-to-end distance consequent on the binding of selected insect counterparts of HMG1 to two DNA fragments, one of 18 bp containing a single dA(2) bulge and a second of 27 bp with two dA(2) bulges. The observed changes are consistent with overall bend angles for the complex of the single HMG domain with one bulge and of two domains with two bulges of approximately 90-100 degrees and approximately 180-200 degrees, respectively. The former value contrasts with an inferred value of 150 degrees reported by Heyduk et al. (1) for the bend induced by a single domain. We also observe that the induced bend angle is unaffected by the presence of the C-terminal acidic region. The DNA bend of approximately 95 degrees observed in the HMG domain complexes is similar in magnitude to that induced by the TATA-binding protein (80 degrees), each monomeric unit of the integration host factor (80 degrees), and the LEF-1 HMG domain (107 degrees). We suggest this value may represent a steric limitation on the extent of DNA bending induced by a single DNA-binding motif.     

Platelet activation studied by fluorescence polarization

Platelet activation was elevated by changes in the fluorescence anisotropy of the sulfhydryl-reactive fluorescent probe, (5-[2-(iodoacetyl) aminacetyl]aminonaphthalene-1-sulfonic acid. The membrane-permeable fluorophore was shown to bind to a multitude of cytoplasmic and membrane proteins. Platelets were stimulated by addition of thrombin, arachidonic acid or ADP under conditions that did not induce aggregation. A sudden increase in the fluorescence anisotropy, r of moderate degree (25-33%) occurred during the first 60 s after exposure of platelets to the aggregating agents and was sustained during the entire period of observation (15-18 min). Phenylmethylsulfonyl thrombin was unable to produce these changes in fluorescence anisotropy. Preincubation of platelets with colchicine reduced r within 30-60 s after platelets were exposed to thrombin. These findings are interpreted as an indication of a general decrease in the 'motional freedom' of the fluorophores and indirectly their ligand molecules.     

Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy

Monomeric red fluorescent proteins (mRFPs) have become indispensable tools for studying protein dynamics, interactions and functions in the cellular environment. Their emission spectrum can be well separated from other fluorescent proteins, and their monomeric structure preserves the natural function of fusion proteins. However, previous photophysical studies of some RFPs have shown the presence of light-induced dark states that can complicate the interpretation of cellular experiments. In this article, we extend these studies to mRFP1, mCherry, and mStrawberry by means of fluorescence correlation spectroscopy and prove that this light-driven intensity flickering also occurs in these proteins. Furthermore, we show that the flickering in these proteins is pH-dependent. Single molecule spectroscopy revealed reversible transitions from a bright to a dark state in several timescales, even up to seconds. Time-resolved fluorescence spectroscopy showed multiexponential decays, consistent with a “loose” conformation. We offer a structural basis for the fluorescence flickering using known crystal structures and point out that the environment of Glu-215 is critical for the pH dependence of the flickering in RFPs. We apply dual-color fluorescence correlation spectroscopy inside live cells to prove that this flickering can seriously hamper cellular measurements if the timescales of the flickering and diffusion are not well separated.     

6-Aminohexanoic acid-plasminogen interactions studied by fluorescence

Circular polarization of luminescence spectra of human plasminogen and of its derivatives were measured in solutions of ligand-free proteins and with saturating amounts of 6-aminohexanoic acid. Spectroscopic changes induced by the ligand reveal similar perturbations of the binding sites in all the protein derivatives. It is concluded that the gross conformational change induced by 6-aminohexanoic acid binding to the native plasminogen involves changes of sterical relations of entire protein domains.     

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high‐speed super‐resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy‐ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.     

The fibronectin binding of salmonellae studied by a passive hemagglutination method]

The study has shown that the fibronectin-binding properties of different Salmonella species can be tested by means of passive hemagglutination with fibronectin-sensitized formulated sheep red blood cells (SRBC). S. enteritidis and S. typhimurium isolated from foodstuffs, as well as the representatives of the species S. kottbus, S. anatum, S. tshiongwe and S. newlands, are capable of binding fibronectin. Fibronectin-binding properties have also been detected in Shigella sonnei, as well as in eubiotic microorganisms Escherichia coli M 17 and Lactobacillus plantarum. In pH ranging from 6.5 to 7.4 salmonellae, shigellae and E. coli agglutinate fibronectin-sensitized SRBC much more effectively in acidic pH values, while changes in pH have been found to produce no essential influence on the fibronectin-binding capacity of Staphylococcus aureus and lactobacilli.     

Slow conformational changes of a Neurospora glutamate dehydrogenase studied by protein fluorescence

1. The NADP-dependent glutamate dehydrogenase of Neurospora crassa undergoes slow reversible structural transitions, with half-times in the order of a few minutes, between active and inactive states. The inactive state of the enzyme, which predominates at pH values below 7.0, has an intrinsic tryptophan fluorescence 25% lower than that of the active state, which predominates at pH values above 7.6. The inactive state can be activated either by an increase in pH or by addition of activators such as succinate. 2. The kinetics of the slow transitions that follow activating and inactivating rapid changes in conditions have been monitored by measurements of protein fluorescence. The results show that the slow reversible conformational change detected by the change in fluorescence is the rate-limiting process for enzyme activation and inactivation. 3. In both directions this conformational change follows apparent first-order kinetics and the rate constant is independent of protein concentration. These kinetics and published measurements of molecular weight are indicative of an isomerization process. 4. In both directions the changes show a large energy of activation and a large positive entropy of activation, consistent with a considerable disturbance of conformation in the transition state. 5. Comparisons of the fluorescence emission spectra of the active and inactive states indicate that the difference in fluorescence is produced by quenching, possibly intramolecular, in the inactive conformation. Iodide ions cause similar quenching. 6. In some mutationally altered forms of the enzyme comparable but modified conformational changes can be followed by protein fluorescence.     

Zinc-secreting Paneth cells studied by ZP fluorescence.

We have used a new family of zinc-specific-responsive fluorescent dyes (ZPs) to study the sequestration and secretion of zinc from Paneth cells, which are located in the bases of the crypts of Lieberkühn within the rat small intestine. Vivid ZP fluorescence zinc staining of Paneth cell secretory granules is seen in both cryostat sections and isolated crypts, providing firm evidence for a pool of labile (rapidly exchangeable) zinc within these cells. We further demonstrate that this ionic zinc pool is secreted under physiological conditions. In vivo stimulation of the small intestine by IP injection of the secretagogue pilocarpine results in discrete zinc staining within the lumens of subsequently isolated crypts, concomitant with a decrease in the zinc staining of Paneth cell granules located within the same crypts. In contrast, the secretion of zinc into the lumens of isolated crypts stimulated in vitro with either carbachol or LPS (lipopolysaccharide) is not observed. However, a distinct change in Paneth cell morphology, suggesting attempted secretion, is seen in response to the direct application of cholinergics but not LPS. These findings suggest that zinc is coreleased with other Paneth cell anti-microbials, and that the intact intestine is necessary for secretion into the crypt lumen.     

Yeast transformation process studied by fluorescence labeling technique

A new method based on fluorescence imaging and flow cytometry was developed to investigate the transformation process of Saccharomyces cerevisiae AY. Yeast and fluorescent-labeled plasmid pUC18 were used as models of cells and DNA molecules, respectively. Binding of DNA molecules to yeast cell surfaces was observed. Factors influencing DNA binding to cell surfaces were investigated. It has been found that poly(ethylene glycol) (PEG) could induce DNA binding to yeast surfaces, while Li(+) showed a weak effect on the binding. When both Li(+) and PEG were used, synergetic effect occurred, resulting in the binding of pUC18 to the surface of more yeast cells compared with that in the presence of PEG or Li(+) only. It was also confirmed that heat shock, Li(+), and PEG all can increase the permeability of yeast cells. This simple method is helpful for understanding the process of yeast transformation and can be used to investigate the interaction of DNA with cell surfaces.     

Viral DNA packaging studied by fluorescence correlation spectroscopy

The DNA packaging machinery of bacteriophage T4 was studied in vitro using fluorescence correlation spectroscopy. The ATP-dependent translocation kinetics of labeled DNA from the bulk solution, to the phage interior, was measured by monitoring the accompanied decrease in DNA diffusibility. It was found that multiple short DNA fragments (100 basepairs) can be sequentially packaged by an individual phage prohead. Fluorescence resonance energy transfer between green fluorescent protein donors within the phage interior and acceptor-labeled DNA was used to confirm DNA packaging. Without ATP, no packaging was observed, and there was no evidence of substrate association with the prohead.     

Microviscosity of togavirus membranes studied by fluorescence depolarization: influence of envelope proteins and the host cell.

The microviscosities of the hydrophobic regions of the membranes of intact Semliki forest and Sindbis viruses grown on BHK-21 cells, of liposomes derived from the extracted viral lipids, and of protease-treated virions were measured by fluorescence depolorization using the fluorescence probe 1, 6-diphenyl-1,3,5-hexatriene. The intact virus membranes were found to have a higher microviscosity than did virus-derived liposomes, indicating the viral envelope proteins contribute to microviscosity. However, protease-treated virus, devoid of protruding spikes but with residual lipophilic peptide tails, was found to have a microviscosity more similar to that of the intact virus than to that of protein-free liposomes. Sindbis virus grown in BHK-21 cells at 37 C had a much higher microviscosity than did Sindbis virus grown on Aedes albopicuts cells at 22 C. Sindbis virus grwon in A. albopictus and BHK-21 cells also gave higher microviscosity values than did the intact host cells. These data indicate that both the virion proteins and the cellular lipids selected during viral growth and maturation contribute to the increased microviscosity of togavirus membranes.