Intrinsic Protein Fluorescence Research Articles

Biological Relevance of the Interaction between Procyanidins and Trypsin: A Multitechnique Approach

The interactions between the digestive protease trypsin type IX-S from porcine pancreas and grape seed procyanidins were monitorized by fluorescence quenching, dynamic light scattering, nephelometry, circular dichroism, and enzymatic inhibition assay. This work reports that the inhibition of trypsin activity by grape seed procyanidins and the respective quenching of intrinsic protein fluorescence are closely related. These two phenomena increase with the molecular weight of the tested procyanidins. The interaction between procyanidins and enzyme was shown to involve a specific interaction as inferred from the fluorescence assays. It was also shown by fluorescence spectroscopy that the binding of procyanidin molecules to the enzyme does not induce significant structural modifications. A relationship between aggregate formation, using dynamic light scattering and nephelometry, and fluorescence quenching was observed with maxima achieved for similar stoichiometric ratios. The binding of procyanidins to trypsin affects only slightly protein structure as seen by circular dichroism.

Indium nanodeposits: A substrate for metal-enhanced fluorescence in the ultraviolet spectral region

We have studied a metallic substrate, composed of indium nanodeposits, for metal-enhanced fluorescence (MEF) in the ultraviolet (UV) spectral region. Indium coated slides were prepared using the thermal vapor deposition technique. Theoretical finite difference time domain simulations and experimental studies show that plasmon enhanced absorption and coupled radiation through the scattering component of the extinction spectra of indium nanoparticles, lie in UV region, and are sensitive to the size and density of the nanoparticles, the thickness of the indium film, and polarity of the medium. The MEF effect, measured for intrinsic protein tryptophan and tyrosine residues, loaded onto indium films of different thickness, changes in a wavelike fashion, reflecting changes in the metal film landscape and, consequently, the chromophores coupling with surface plasmons. Indium films also significantly enhance intrinsic fluorescence of proteins themselves [bovine serum albumin]. In this case the wavelength dependence of MEF shows different emission enhancements of protein Tyr and Trp residues. Subsequently, indium-enhanced intrinsic protein fluorescence in the UV spectral region can be of great potential importance for quantitation assays as well as for the labeless detection of biomolecules in the biosciences.

Study of the Interaction of Pancreatic Lipase with Procyanidins by Optical and Enzymatic Methods

The interactions between porcine pancreatic lipase (PL) and grape seed procyanidins were studied by an enzymatic assay, fluorescence quenching, nephelometry, and dynamic light scattering (DLS). An inhibitory effect of grape seed procyanidins on lipase hydrolytic activity was found. Both the inhibition of lipase activity by procyanidins and the respective quenching of intrinsic protein fluorescence increased with the average degree of polymerization of the tested procyanidins. The association between procyanidins and enzyme involves a specific interaction as inferred from the fluorescence assays despite not changing significantly the tertiary structure of the protein. For all tested procyanidins it was shown, both by DLS and by nephelometry, that an increase in aggregation occurs up to a stoichiometric maximum after which further procyanidin addition causes a decrease in aggregation of aggregates. The maximum size of aggregates was shown to be closely related to the maximum overall aggregation. It was also shown that the inhibition of enzyme activity is to a large extent independent of the formation of aggregates.

Kinetics of Surfactant-induced Aggregation of Lysozyme Studied by Fluorescence Spectroscopy

The study of protein conformational changes in the presence of surfactants and lipids is important in the context of protein folding and misfolding. In the present study, we have investigated the mechanism of the protein conformational change coupled with aggregation leading to size growth of Hen Egg White Lysozyme (HEWL) in the presence of an anionic detergent such as sodium dodecyl sulphate (SDS) in alkaline pH. We have utilized intrinsic protein fluorescence (tryptophan) and extrinsic fluorescent reporters such as 8-anilinonaphthalene-1-sulfonic acid (ANS), dansyl and fluorescein to follow the protein conformational change in real-time. By analyzing the kinetics of fluorescence intensity and anisotropy of multiple fluorescent reporters, we have been able to delineate the mechanism of surfactant-induced aggregation of lysozyme. The kinetic parameters reveal that aggregation proceeds with an initial fast-phase (conformational change) followed by a slow-phase (self-assembly). Our results indicate that SDS, below critical micelle concentration, induces conformational expansion that triggers the aggregation process at a micromolar protein concentration range.

Temperature-Responsive Release of Cortisol from Its Binding Globulin: A Protein Thermocouple

Only 5% of circulating cortisol is active and unbound to carrier proteins. Because cortisol levels vary rapidly due to the pulsatile nature of cortisol secretion, the dynamics of cortisol binding are critical determinants of tissue levels of free cortisol and consequent hormonal signaling. The major glucocorticoid carrier protein is corticosteroid binding globulin (CBG), a member of the serpin family that undergoes conformational changes to bind and release hormones. This mechanism has been noted to be temperature responsive, and we have now investigated the effects of temperature on the binding of human CBG to both cortisol and progesterone. Recombinant human CBG was synthesized and used for binding studies with cortisol and progesterone between 34 and 43 C. Binding was monitored by recording the change in intrinsic protein fluorescence. Binding of the steroids to the other major carrier, serum albumin, was measured in a similar manner. There was no effect of temperature on the interaction between human serum albumin and either cortisol or progesterone. The association of both cortisol and progesterone with CBG is more than three orders of magnitude greater than that with HSA, and this interaction was extremely responsive to changes in temperature. The affinity of both cortisol and progesterone for CBG drops approximately 16-fold as temperature increases from 35 to 42 C. This study clearly shows that even within the clinically relevant range of temperatures found in humans, CBG acts as a protein thermocouple that is exquisitely sensitive to temperature change and will release cortisol in response to fever or external sources of heat. This has major implications for our understanding of cortisol regulation in febrile patients.

Kinetics of ATP and TNP-ATP Binding to the Active Site of CheA from Thermotoga maritima

The mechanism of nucleotide binding to the active site of Thermotoga maritima CheA was investigated using stopped-flow fluorescence experiments that monitored binding of ATP and TNP-ATP to the catalytic domain (P4) of CheA that had been engineered to include a tryptophan residue as a fluorescent reporter group at the active site (P4(F487W)). Rapid decreases in protein intrinsic fluorescence and increases in TNP-ATP fluorescence were observed during binding reactions, and time courses were analyzed to define the kinetic mechanisms for ATP and TNP-ATP binding. This analysis indicated that binding of ATP(Mg(2+)) to P4(F487W) involves a single reversible step with a k(on) of 0.92 +/- 0.09 microM(-1) s(-1), a k(off) of 1.9 +/- 0.4 s(-1), and a K(d) of 1.5-2.1 microM (all values determined at 4 degrees C). Binding of TNP-ATP(Mg(2+)) to P4(F487W) involves a more complicated mechanism, requiring at least three sequential steps. Computer simulations and nonlinear regression analysis were used to estimate the rate constants of the forward and reverse reactions for each of the three steps in the reaction scheme [Formula: see text] Similar analysis indicated that an alternative reaction scheme, involving a rate-limiting conformational change in P4 prior to TNP-ATP binding, did an equally good job of accounting for all of the kinetics results:[Formula: see text] In both models, steps 2 and 3 have slow reversal rates that contribute to the high affinity of the active site for TNP-ATP (K(d) = 0.015 microM). These results highlight the dramatic effect of the TNP moieties on CheA-nucleotide interactions, and they provide the first detailed information about the kinetic mechanism underlying interaction of a protein histidine kinase with this tight-binding inhibitor.

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10(8) molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

β-Lactoglobulin/Folic Acid Complexes: Formation, Characterization, and Biological Implication

Beta-lactoglobulin (beta-LG), the major whey protein in bovine milk, binds to a wide range of compounds. Folic acid (FA) is a synthetic form of the B group vitamin known as folates, which are essential cofactors for a variety of physiological processes. The interaction of beta-LG with FA was studied using fluorescence spectroscopy to determine the FA binding constant and mode and the influence of the protein on FA photodegradation. At < or = 20 microM FA, which may be the critical self-association concentration, the binding constant and number are 2.0 (+/-0.6) x 10(6) M(-1) and 1.30 (+/-0.03) when excited at 280 nm and 4.3 (+/-2.2) x 10(5) M(-1) and 1.17 (+/-0.04) at 295 nm, as determined by protein intrinsic fluorescence. FA binds to the surface of beta-LG, possibly in the groove between the alpha-helix and the beta-barrel. Fluorescence analysis of the pterin portion of FA shows that complexation with beta-LG improves FA photostability. It is suggested that beta-LG complexes could be used as an effective carrier of FA in functional foods.

Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone

ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 degrees C but not at 4 degrees C. Neither MgADP nor MgATP-gamma-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle.

LCP-Tm: An Assay to Measure and Understand Stability of Membrane Proteins in a Membrane Environment

Structural and functional studies of membrane proteins are limited by their poor stability outside the native membrane environment. The development of novel methods to efficiently stabilize membrane proteins immediately after purification is important for biophysical studies, and is likely to be critical for studying the more challenging human targets. Lipidic cubic phase (LCP) provides a suitable stabilizing matrix for studying membrane proteins by spectroscopic and other biophysical techniques, including obtaining highly ordered membrane protein crystals for structural studies. We have developed a robust and accurate assay, LCP-Tm, for measuring the thermal stability of membrane proteins embedded in an LCP matrix. In its two implementations, protein denaturation is followed either by a change in the intrinsic protein fluorescence on ligand release, or by an increase in the fluorescence of a thiol-binding reporter dye that measures exposure of cysteines buried in the native structure. Application of the LCP-Tm assay to an engineered human β2-adrenergic receptor and bacteriorhodopsin revealed a number of factors that increased protein stability in LCP. This assay has the potential to guide protein engineering efforts and identify stabilizing conditions that may improve the chances of obtaining high-resolution structures of intrinsically unstable membrane proteins.

Structural and functional characterization of the core single‐stranded DNA‐binding domain of purine‐rich element binding protein B (Purβ)

Purβ is a sequence‐specific single‐stranded DNA (ssDNA)‐binding protein that represses smooth muscle α‐actin gene transcription in stress‐activated fibroblasts and smooth muscle cells via cooperative interaction with two sites in the purine‐rich strand of a 5′ enhancer element (dubbed PE32‐F). To better understand how the modular structure of Purβ dictates its specific ssDNA‐binding function, we evaluated the interaction of purified recombinant Purβ with PE32‐F using spectroscopic and biochemical approaches. Results of intrinsic protein fluorescence and circular dichroism analyses pointed to a ssDNA‐induced change in protein conformation. Consistent with this interpretation, Purβ was protected from proteolysis when complexed with PE32‐F. Limited tryptic digestion of Purβ liberated a core ~30 kDa fragment spanning residues 29‐305 as determined by mass spectrometry. Size exclusion chromatography indicated that the isolated core fragment retains the ability to dimerize. Quantitative ssDNA‐binding assays revealed that the core domain binds to PE32‐F with similar specificity but increased affinity compared to full‐length Purβ and a truncated 37‐263 mutant absent a putative amphipathic helix‐forming module. These findings provide new insight into the structural basis for the specific and high affinity binding of Purβ to ssDNA. NIH R01 HL054281, T32 HL007594, P20 RR16462.

A stopped‐flow kinetic analysis of substrate binding and catalysis by Escherichia coli RNA polymerase

To test the hypothesis that the binding of the correct NTP to a pre‐insertion site triggers rapid pyrophosphate (PPi) release followed by entry of the NTP into the active site for incorporation, we used the intrinsic protein fluorescence of the core polymerase in well‐defined elongation complexes to monitor nucleotide binding and incorporation. The increase in fluorescence in each case could be fitted by a single exponential. Plots of the observed first order rate constants versus NTP concentration were hyperbolic. This is consistent with a mechanism involving rapid binding followed by a slow conformational change. In the case of UMP incorporation, the value of the rate constant for the conformational change was 590±60 s−1 and the value of the apparent dissociation constant for UTP was 8.6±3.8 μM; in the case of AMP incorporation, the corresponding values were 520±50 s−1 and 15.7±5.6 μM. The rate constant for PPi release in the presence of the next correct nucleotide for incorporation was reported previously to be 587±178 s−1. The coincidence between the rate constants for the conformational change and PPi release is consistent with the postulated mechanism. NSF Grant #211369

Developing Reporter Systems to Monitor the Structural Dynamics of MutS

DNA mismatch repair (MMR) is an evolutionarily conserved process that locates and repairs post‐replicative errors in nascent DNA and is critical for maintaining the genome integrity. The first step in the MMR pathway is recognition of the base‐base mismatch/insertion deletion loop (IDL) by MutS in prokaryotes and the MutS homologues, Msh2‐Msh6 and Msh2‐Msh3, in eukaryotes. While there is a significant body of literature concerning DNA‐binding and ATPase activities of MutS, information on MutS conformational changes involved in recognizing an array of non‐Watson‐Crick DNA structures is lacking. Here we present two fluorescence reporter assays developed to examine T. aquaticus MutS conformational changes and better understand the relationship between its structure/dynamics and function. Two mutant proteins were prepared: 1) F39, which contacts DNA, was replaced with tryptophan to monitor intrinsic protein fluorescence during the reaction; 2) M88 in the DNA binding domain was replaced by cysteine and labeled with a thiol reactive fluorophore that is sensitive to protein conformation. We are currently measuring changes in T. aquaticus MutS conformation on mismatch/IDL binding in equilibrium and in real time by transient kinetic analysis.

Mechanism and Specificity of a Symmetrical Benzimidazolephenylcarboxamide Helicase Inhibitor

This study examines the effects of 1-N,4-N-bis[4-(1H-benzimidazol-2-yl)phenyl]benzene-1,4-dicarboxamide ((BIP)(2)B) on the NS3 helicase encoded by the hepatitis C virus (HCV). Molecular beacon-based helicase assays were used to show that (BIP)(2)B inhibits the ability of HCV helicase to separate a variety of RNA and DNA duplexes with half-maximal inhibitory concentrations ranging from 0.7 to 5 microM, depending on the nature of the substrate. In single turnover assays, (BIP)(2)B only inhibited unwinding reactions when it was preincubated with the helicase-nucleic acid complex. (BIP)(2)B quenched NS3 intrinsic protein fluorescence with an apparent dissociation constant of 5 microM, and in the presence of (BIP)(2)B, HCV helicase did not appear to interact with a fluorescent DNA oligonucleotide. In assays monitoring HCV helicase-catalyzed ATP hydrolysis, (BIP)(2)B only inhibited helicase-catalyzed ATP hydrolysis in the presence of intermediate concentrations of RNA, suggesting RNA and (BIP)(2)B compete for the same binding site. HCV helicases isolated from various HCV genotypes were similarly sensitive to (BIP)(2)B, with half-maximal inhibitory concentrations ranging from 0.7 to 2.4 microM. (BIP)(2)B also inhibited ATP hydrolysis catalyzed by related helicases from Dengue virus, Japanese encephalitis virus, and humans. (BIP)(2)B appeared to bind the HCV and human proteins with similar affinity (K(i) = 7 and 8 microM, respectively), but it bound the flavivirus proteins up to 270 times more tightly. Results are discussed in light of a molecular model of a (BIP)(2)B-HCV helicase complex, which is unable to bind nucleic acid, thus preventing the enzyme from separating double-stranded nucleic acid.

Structure and stability of D-galactose/D-glucose-binding protein. The role of D-glucose binding and Ca ion depletion

The effects of guanidine hydrochloride (GdnHCl) on the structure and stability of the D-galactose/D-glucose-binding protein from Escherichia coli (GGBP) and its complex with D-glucose (GGBP/Glc) were investigated by intrinsic protein fluorescence and far-UV circular dichroism (CD). The role of calcium in the stability of the protein structure was also studied. It was shown that the processes of GGBP and GGBP/Glc unfolding induced by GdnHCl followed one-step reversible denaturation mechanism. The obtained data showed that the binding of glucose to GGBP resulted in an increase of the protein stability towards the actions of the GdnHCl which made protein unfolding more cooperative. The stabilities of GGBP alone, GGBP in the presence of glucose, GGBP-depleted calcium (GGBP-Ca), and GGBP/Glc-depleted calcium (GGBP/Glc-Ca) were characterized by difference of Gibbs free energies.

Structure and stability of D-galactose/D-glucose-binding protein. The role of D-glucose binding and Ca ion depletion

The effects of guanidine hydrochloride (GdnHCl) on the structure and stability of the D-galactose/D-glucose-binding protein fromEscherichia coli(GGBP) and its complex with D-glucose (GGBP/Glc) were investigated by intrinsic protein fluorescence and far-UV circular dichroism (CD). The role of calcium in the stability of the protein structure was also studied. It was shown that the processes of GGBP and GGBP/Glc unfolding induced by GdnHCl followed one-step reversible denaturation mechanism. The obtained data showed that the binding of glucose to GGBP resulted in an increase of the protein stability towards the actions of the GdnHCl which made protein unfolding more cooperative. The stabilities of GGBP alone, GGBP in the presence of glucose, GGBP-depleted calcium (GGBP-Ca), and GGBP/Glc-depleted calcium (GGBP/Glc-Ca) were characterized by difference of Gibbs free energies.

10.1007/s11171-008-2004-0

The encoding sequence of the pore-forming OmpF-like protein from the Yersinia pseudotuberculosis outer membrane was cloned and expressed in Escherichia coli cells. Conditions for isolation and refolding of recombinant monomer and porin trimer were selected. Their spatial structures were characterized by the intrinsic protein fluorescence and CD spectroscopy. It was shown that recombinant porins are similar in the composition of secondary structure elements to isolated porins, but have a considerably less compact tertiary structure. The pore-forming activities of the recombinant proteins are similar to those of Y. pseudotuberculosis native porins.

Application of Phasor Plots to Analysis of Fluorophore Heterogeneity, Excited State Reactions and Protein Conformations

Phasor plots provide a simple graphical method to visualize and quantify time resolved fluorescence data, obtained using either frequency or time domain methods, independent of model constraints. Using the phase and modulation approach, the phasor plot converts raw data at a single frequency to a vector. Single exponential decays appear on the universal circle (semicircle with radius 0.5 and center 0.5, 0) whereas decays due to multiple exponentials appear as points inside the universal circle. This method has been successfully applied to fluorescence lifetime imaging microscopy (FLIM) wherein the data are typically collected at only one frequency. Applications of phasor plots in FLIM studies have, to date, been largely limited to FRET studies in cells. We have extended the application of phasor plots to several in vitro systems. Specifically, we have analyzed frequency-domain data of binary and tertiary mixtures of non-interacting, monoexponential-decay fluorophores and intrinsic protein fluorescence using the phasor plot approach. Phasor points from binary mixtures of varying composition lie along the line connecting the individual component points on the universal circle, while tertiary mixture points fall in a triangle between the individual vectors. Molecular interactions such as protein dissociation, protein-ligand interaction, denaturation, and energy transfer resulted in changes in the position of the vector point allowing for a rapid, graphical representation of these complex reactions. Data at a single frequency may be recorded rapidly allowing resolution of kinetic processes that would be difficult to monitor using complete multifrequency approaches. The combined results demonstrate the value of the phasor plot method to in vitro lifetime analysis. This work was supported in part by a grant from Allergan, Inc.

Photophysics and photochemistry of dyes bound to human serum albumin are determined by the dye localization

The photophysics and photochemistry of rose bengal (RB) and methylene blue (MB) bound to human serum albumin (HSA) have been investigated under a variety of experimental conditions. Distribution of the dyes between the external solvent and the protein has been estimated by physical separation and fluorescence measurements. The main localization of protein-bound dye molecules was estimated by the intrinsic fluorescence quenching, displacement of fluorescent probes bound to specific protein sites, and by docking modelling. All the data indicate that, at low occupation numbers, RB binds strongly to the HSA site I, while MB localizes predominantly in the protein binding site II. This different localization explains the observed differences in the dyes' photochemical behaviour. In particular, the environment provided by site I is less polar and considerably less accessible to oxygen. The localization of RB in site I also leads to an efficient quenching of the intrinsic protein fluorescence (ascribed to the nearby Trp residue) and the generation of intra-protein singlet oxygen, whose behaviour is different to that observed in the external solvent or when it is generated by bound MB.

Kinetic Mechanism for the Binding of eIF4F and Tobacco Etch Virus Internal Ribosome Entry Site RNA

The wheat germ eukaryotic translation initiation factor (eIF) 4F binds tightly to the mRNA internal ribosome entry site (IRES) of tobacco etch virus (TEV) to promote translation initiation. When eIF4F is limiting, TEV is preferentially translated compared with host cell mRNA. To gain insight into the dynamic process of protein synthesis initiation and the mechanism of binding, the kinetics of eIF4F binding to TEV IRES were examined. The association rate constant (k(on)) and dissociation rate constant (k(off)) for eIF4F binding to IRES were 59 +/- 2.1 micro s(-1) and 12.9 +/- 0.3 s(-1), respectively, comparable with the rates for capped RNA. Binding of eIF4E or eIF4F to the cap of mRNA is the rate-limiting step for initiation of cap-dependent protein synthesis. The concentration dependence of the reactions suggested a simple one-step association mechanism. However, the association rate was reduced more than 10-fold when KCl concentration was increased from 50 to 300 mm, whereas the dissociation rate constant was increased 2-fold. The addition of eIF4B and poly(A)-binding protein enhanced the association rate of eIF4F approximately 3-fold. These results suggest a mechanism where eIF4F initially binds electrostatically, followed by a conformational change to further stabilize binding. Poly(A)-binding protein and eIF4B mainly affect the eIF4F/TEV association rate. These results demonstrate the first direct kinetic measurements of translation initiation factor binding to an IRES.