Changes In Protein Fluorescence Research Articles

Evaluation of Phenol Sulfotransferase (SULT1A1) Ligand Binding Order

Cytosolic sulfotransferases (SULTs) modify and regulate many endogenous and xenobiotic compounds. There are some disagreements regarding the binding pattern of their nucleotide (PAPS, PAP) and acceptor site ligands. Here we present an investigation of the homodimeric bovine phenol sulfotransferase (bSULT1A1) using several methods not dependent on catalytic turnover. Binding order was not determinable from intrinsic protein fluorescence changes in response to PAP(S), 7-hydroxycoumarin (7-HC) or pentachlorophenol (PCP). Equilibrium dialysis showed no binding of 7-HC to apo-bSULT1A1 up to 100 µM ligand, but inclusion of PAP induced biphasic binding of 7-HC. Isothermal titration calorimetry revealed exothermic half-sites binding of PAP to the dimeric protein. No binding of 7-HC to apo-SULT1A1 was detected, but SULT1A1:PAP also bound 7-HC at 0.5 per subunit. Differential scanning fluorimetry failed to show thermal stabilization of protein by excess 7-HC or PCP, whereas inclusion of PAP increased T_m dramatically. Susceptibility of bSULT1A1 to cleavage by chymotrypsin was not affected by excess 7-HC, but enzyme with PAP ± 7-HC resisted proteolysis. Excess 7-HC did not change protein cysteine reactivity with DTNB, whereas PAP impeded the fast phase of reaction attributable to C168. These observations indicate an ordered binding scheme for bSULT1A1. Testing other SULT ligand binding patterns with these methods is suggested.

Changes in Protein Fluorescence in a Lipid-Protein Co-oxidizing Oleogel.

High and low levels of lipid-induced protein oxidation (tuned by the addition of 0%-8.4% water) were investigated in oleogels, using excitation-emission matrix (EEM) fluorescence spectroscopy, coupled with a partial least-squares (PLS) regression and lipid hydroperoxide data. In high-level oxidation models, the intrinsic tryptophan fluorescence decreased and the emission maxima increased from 352.5 to 356.0 nm indicating the presence of protein modifications, which was further supported by size-exclusion chromatography. PLS recognized 3 latent components, with several excitation-emission points of interest. These apparent compounds include a region associated with radical mediated protein modifications (approximately 325 and 410 nm), lipid oxidation product adducts (approximately 350 nm and 420-425 nm), and malondialdehyde adducts (approximately 375 and 425 nm). The separate evaluation of these apparent compounds, at a 420 nm emission, indicated that lipid oxidation promotes protein lipid adduct fluorescence at high water levels, rather than radical mediated protein fluorescence.

Comparison of the functional properties of trimeric and monomeric CaiT of Escherichia coli

Secondary transporters exist as monomers, dimers or higher state oligomers. The significance of the oligomeric state is only partially understood. Here, the significance of the trimeric state of the L-carnitine/γ-butyrobetaine antiporter CaiT of Escherichia coli was investigated. Amino acids important for trimer stability were identified and experimentally verified. Among others, CaiT-D288A and -D288R proved to be mostly monomeric in detergent solution and after reconstitution into proteoliposomes, as shown by blue native gel electrophoresis, gel filtration, and determination of intermolecular distances. CaiT-D288A was fully functional with kinetic parameters similar to the trimeric wild-type. Significant differences in amount and stability in the cell membrane between monomeric and trimeric CaiT were not observed. Contrary to trimeric CaiT, addition of substrate had no or only a minor effect on the tryptophan fluorescence of monomeric CaiT. The results suggest that physical contacts between protomers are important for the substrate-induced changes in protein fluorescence and the underlying conformational alterations.

Differential Kinetics of Two-Cysteine Peroxiredoxin Disulfide Formation Reveal a Novel Model for Peroxide Sensing.

Two-cysteine peroxiredoxins (Prx) have a three-step catalytic cycle consisting of (1) reduction of peroxide and formation of sulfenic acid on the enzyme, (2) condensation of the sulfenic acid with a thiol to form disulfide, also known as resolution, and (3) reduction of the disulfide by a reductant protein. By following changes in protein fluorescence, we have studied the pH dependence of reaction 2 in human peroxiredoxins 1, 2, and 5 and in Salmonella typhimurium AhpC and obtained rate constants for the reaction and p Ka values of the thiol and sulfenic acid involved for each system. The observed reaction 2 rate constant spans 2 orders of magnitude, but in all cases, reaction 2 appears to be slow compared to the same reaction in small-molecule systems, making clear the rates are limited by conformational features of the proteins. For each Prx, reaction 2 will become rate-limiting at some critical steady-state concentration of H2O2 producing the accumulation of Prx as sulfenic acid. When this happens, an alternative and faster-resolving Prx (or other peroxidase) may take over the antioxidant role. The accumulation of sulfenic acid Prx at distinct concentrations of H2O2 is embedded in the kinetic limitations of the catalytic cycle and may constitute the basis of a H2O2-mediated redox signal transduction pathway requiring neither inactivation nor posttranslational modification. The differences in the rate constants of resolution among Prx coexisting in the same compartment may partially explain their complementation in antioxidant function and stepwise sensing of H2O2 concentration.

Comparative Studies on the Interaction Between the Medicine Small Molecule with Pepsin by Fluorescence Quenching Spectroscopy and Improved Spectroscopy

The binding mechanism between cefetamet pivoxil (CFP) and pepsin (PEP) at different temperatures (298 K, 303 K, 310 K) was investigated by the classical fluorescence spectroscopy with focus on the fluorescence change of protein, as well as the improved spectroscopy with focus on the fluorescence changes of the resonance light scattering of small molecule drugs. The results showed that the main quenching mode of PEP-CFP was static quenching. The value of n was approximately equal to 1 which indicating that there was only one binding site in the interaction between PEP and CFP and the Hill coefficient was about 1 which indicating that there was no cooperative between the receptor PEP and ligand CFP. The binding constants of the PEP-CFP system obtained by the improved spectroscopic method were two orders of magnitude larger than that of the traditional fluorescence spectroscopy, which showed that the study of the small drug molecule was more practical and reasonable. The rationality of the experimental results obtained was verified by ultraviolet absorption spectroscopy.

A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

A possible biochemical basis for fructose-induced inhibition of embryo development in Norway spruce (Picea abies).

Sugars play an important role in various physiological processes during plant growth and development; however, the developmental roles and regulatory functions of hexoses other than glucose are still largely unclear. Recent studies suggest that blocked embryo development in Norway spruce (Picea abies (L.) Karst) is associated with accumulation of fructose. In the present study, the potential biochemical regulatory mechanism of glucose and fructose was studied during development of somatic embryos of Norway spruce from pro-embryogenic masses to mature embryos. The changes in protein fluorescence, a marker of the Maillard reaction, were monitored in two cell lines of Norway spruce that were grown on media containing sucrose (control), glucose or fructose. Manual time-lapse photography showed that growth of embryogenic cultures on medium containing sucrose was characterized by normal development of mature embryos whereas the embryogenic cultures that were grown on media containing glucose or fructose did not develop mature embryos. The biochemical analyses of embryogenic samples collected during embryo development showed that: (i) the content of glucose and fructose in the embryogenic cultures increased significantly during growth on each medium, respectively; (ii) the accumulation of Maillard products in the embryogenic cultures was highly correlated with the endogenous content of fructose but not glucose; and (iii) the embryogenic cultures grown on fructose displayed the highest protein carbonyl content and DNA damage whereas the highest content of glutathione was recorded in the embryogenic cultures that had grown on sucrose. Our data suggest that blocked development of embryos in the presence of fructose may be associated with the Maillard reaction.

FXYD Proteins Stabilize Na,K-ATPase: AMPLIFICATION OF SPECIFIC PHOSPHATIDYLSERINE-PROTEIN INTERACTIONS

FXYD proteins are a family of seven small regulatory proteins, expressed in a tissue-specific manner, that associate with Na,K-ATPase as subsidiary subunits and modulate kinetic properties. This study describes an additional property of FXYD proteins as stabilizers of Na,K-ATPase. FXYD1 (phospholemman), FXYD2 (γ subunit), and FXYD4 (CHIF) have been expressed in Escherichia coli and purified. These FXYD proteins associate spontaneously in vitro with detergent-soluble purified recombinant human Na,K-ATPase (α1β1) to form α1β1FXYD complexes. Compared with the control (α1β1), all three FXYD proteins strongly protect Na,K-ATPase activity against inactivation by heating or excess detergent (C(12)E(8)), with effectiveness FXYD1 > FXYD2 ≥ FXYD4. Heating also inactivates E(1) ↔ E(2) conformational changes and cation occlusion, and FXYD1 protects strongly. Incubation of α1β1 or α1β1FXYD complexes with guanidinium chloride (up to 6 m) causes protein unfolding, detected by changes in protein fluorescence, but FXYD proteins do not protect. Thus, general protein denaturation is not the cause of thermally mediated or detergent-mediated inactivation. By contrast, the experiments show that displacement of specifically bound phosphatidylserine is the primary cause of thermally mediated or detergent-mediated inactivation, and FXYD proteins stabilize phosphatidylserine-Na,K-ATPase interactions. Phosphatidylserine probably binds near trans-membrane segments M9 of the α subunit and the FXYD protein, which are in proximity. FXYD1, FXYD2, and FXYD4 co-expressed in HeLa cells with rat α1 protect strongly against thermal inactivation. Stabilization of Na,K-ATPase by three FXYD proteins in a mammalian cell membrane, as well the purified recombinant Na,K-ATPase, suggests that stabilization is a general property of FXYD proteins, consistent with a significant biological function.

Kinetics of Mouse Alpha-Myosin S1 a Fast Myosin

The fast skeletal muscle myosins and the slow/β-cardiac myosins have been well described but few kinetic studies of the α cardiac myosin have been presented. Here we show that the mouse alpha cardiac myosin has more in common with fast skeletal type myosin than the slower B-type myosin. The data for individual rate constant for mouse α myosin S1 are compared to our published values for the rabbit fast muscle and slow/β myosin S1. The ADP affinity for acto.S1 is much weaker (250 µM) than beta-S1 (10 µM) and similar to that of fast rabbit muscle S1 (120 μM). The rate constant for ADP release is too fast to measure for mouse α -S1 and rabbit fast muscle S1 but 63 s−1 for the rabbit β-S1. For all myosins affinity of actin form myosin is reduced by ADP and the affinity of ADP for S1 is reduced by actin. This thermodynamic coupling ratio is 10 for β-S1 but ∼50-100 for fast muscle S1 and α-S1. The ATP hydrolysis step rate constant estimated from the protein fluorescence changes is 150 s−1 similar tor fast muscle S1 (120 s−1) and faster that β-S1 (21 s−1). These values suggest that the ADP release from AMD and the hyrdolysis step are changed in parallel and this is consistent with the hydrolysis step having a role in defining the lifetime of the detached state (M.ATP) while ADP release controls the lifetime of the attached state. Thus the duty ratio can remain similar while the cycle time is altered. All other values assayed show no major difference between the three isoforms of myosin. Overall despite the greater sequence similarity between α and β cardic S1 the α-S1 shares kinetic properties with the fast muscle S1.

Influence of photodynamic effect on biological activity of PBR–PP complexes

The aim of this study was to examine the influence of photodynamic effect on biological activity of PBR–PP complexes. These measurements were performed in pH dependent environment. Constant concentration of solubilized receptor was titrated with increasing concentration of porphyrins (PPIX, Hp, PP(Arg) 2, Hp(Arg) 2, PP(Gly) 2, PP(Ala) 2, PP(Ser) 2, PP(Phe) 2) and binding constants were calculated. PBP–PP mixtures were illuminated with 3 J, 5 J or 10 J of blue light and changes in protein fluorescence was recorded. Experimental data were fitted to weak and strong binding models. As a result for all derivatives weak binding model was the best fitted. The strongest binding showed PPIX in pH 7.4 and with pH drop binding constants showed greater values for all examined derivatives. Out of amino acid derivatives the strongest binding was noticed for PP(Gly) 2 and PP(Phe) 2 and for the last one pH influence was not observed.

5-Aminolaevulinic acid dehydratase: characterization of the alpha and beta metal-binding sites of the Escherichia coli enzyme.

The alpha and beta metal-binding sites of 5-aminolaevulinic acid dehydratase (ALAD) (porphobilinogen synthase, EC 4.2.1.24) from Escherichia coli were investigated to determine the function of each metal ion and the role of the reactive cysteines in metal binding. Occupancy of the alpha site by Zn2+ restored virtually all catalytic activity to the inactive metal-depleted ALAD (apoALAD). Occupancy of the alpha site by Co2+ also yielded an active enzyme and resulted in a charge-transfer band indicative of a single cysteine amongst the metal ligands. Subsequent labelling of this cysteine residue with 14C-labelled N-ethylmaleimide, followed by peptide analysis, indicated the involvement of Cys-130. The metal ion at the alpha site is thought to be essential for binding of the second molecule of substrate at the A substrate-binding site that forms the acetic acid side of the product, porphobilinogen. Binding of Zn2+ to the beta site restored little activity if the alpha site was unfilled. Metal ion binding to the beta site could be monitored by following the change in protein fluorescence with Zn2+ titration of apoALAD at pH 6. A conformational change upon beta site occupancy may explain why binding of Mg2+ at the alpha site can occur only if Zn2+ is bound at the beta site. The binding of Co2+ at the beta site produced an inactive enzyme that exhibited a charge-transfer band indicative of at least three cysteine ligands.

Specific Protein Domains Mediate Cooperative Assembly of HuR Oligomers on AU-rich mRNA-destabilizing Sequences

The RNA-binding factor HuR is a ubiquitously expressed member of the Hu protein family that binds and stabilizes mRNAs containing AU-rich elements (AREs). Hu proteins share a common domain organization of two tandemly arrayed RNA recognition motifs (RRMs) near the N terminus, followed by a basic hinge domain and a third RRM near the C terminus. In this study, we engineered recombinant wild-type and mutant HuR proteins lacking affinity tags to characterize their ARE-binding properties. Using combinations of electrophoretic mobility shift and fluorescence anisotropy-based binding assays, we show that HuR can bind ARE substrates as small as 13 nucleotides with low nanomolar affinity, but forms cooperative oligomeric protein complexes on ARE substrates of at least 18 nucleotides in length. Analyses of deletion mutant proteins indicated that RRM3 does not contribute to high affinity recognition of ARE substrates, but is required for cooperative assembly of HuR oligomers on RNA. Finally, the hinge domain between RRM2 and RRM3 contributes significant binding energy to HuR.ARE complex formation in an ARE length-dependent manner. The hinge does not enhance RNA-binding activity by increased ion pair formation despite extensive positive charge within this region, and it does not thermodynamically stabilize protein folding. Together, the results define distinct roles for the HuR hinge and RRM3 domains in formation of cooperative HuR.ARE complexes in solution.

Charge promiscuity in the metal ion activation of S‐Adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, Tb3+, Gd3+, Eu3+, in addition to the known divalent cation activators Mg2+, Mn2+ and Ca2+. All of the activating cations provide kcat values within an order of magnitude of one another, suggesting that role of the cation does not predominantly lie in the rate limiting step of the reaction. The KM for AdoMet varies less than 4-fold with the different metal ion activators. Cation concentrations for half maximal activation decrease with each increment of increase in the cation charge, from ~ 300 mM with Li+ to ~ 2 μM with trivalent lanthanide ions. The exchange inert Co(NH3)63+ cation activates in the presence of EDTA and to a higher level than Co2+ (NH4+ does not activate) indicating that direct metal coordination to the substrate is not required for activation Equilibrium binding studies monitoring protein fluorescence changes show that K+, Zn2+ and Rh3+interact with the protein, but these cations do not activate. The binding of metal ion activators and enzyme activation are both cooperative with Hill coefficients as large as 4, indicating that the binding of one equivalent of some cations can activate the entire (αβ )4 enzyme. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase. Supported by NIH grant GM31186

Mechanical perturbation-induced fluorescence change of green fluorescent protein

The force curve measurement mode of the atomic force microscope (AFM) is a powerful experimental technique in biotechnology. However, it is more effective if the spectroscopic properties of the biomolecule in the contact area can be simultaneously measured. Thus, we developed a confocal laser scanning microscope/AFM system. In this study, we simultaneously measured the fluorescence spectra of green fluorescent protein with the application of an external force in order to investigate the stability and dynamics of the β-barrel structure. Consequently, the fluorescence was quenched by applying both compression and extension forces and the quenching efficiencies differed in each case.

Folding Kinetics of an α Helical Membrane Protein in Phospholipid Bilayer Vesicles

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.

3P299 AFMプローブで加えた力学的摂動によるGFPの発光変化の観察(計測)

3P299 AFMプローブで加えた力学的摂動によるGFPの発光変化の観察(計測)

Unfolding and refolding of porcine odorant binding protein in guanidinium hydrochloride: equilibrium studies at neutral pH

Unfolding and refolding studies on porcine odorant binding protein (pOBP) have been performed at pH 7 in the presence of guanidinium hydrochloride (GdnHCl). Unfolding, monitored by following changes of protein fluorescence and circular dichroism (CD), was found to be a reversible process, in terms of recovered structure and function. The equilibrium transition data were fitted by a simple two-state sigmoidal function of denaturant concentration and the thermodynamic folding parameters, derived from the two techniques, were very similar (average values: C 1/2≈2.4 M, m≈2 kcal mol −1 M −1, Δ G unf,w 0≈4.7 kcal mol −1). The transition was independent of protein concentration, indicating that only monomeric species are involved. Only a minor protective effect by the fluorescent ligand 1-amino-anthracene (AMA) against protein unfolding was detected, whereas dihydromyrcenol (DHM) stabilised the protein to a larger extent (Δ C 1/2≈0.5 M). Refolding was complete, when the protein, denatured with GdnHCl, was diluted with buffer. On the other hand, refolding by dialysis was largely prevented by concomitant aggregation. The present results on pOBP are compared with those on bovine OBP (bOBP) [Biochim. Biophys. Acta 1599 (2002) 90], where subunit folding is accompanied by domain swapping. We finally suggest that the generally observed two-state folding of many lipocalins is probably favoured by their β-barrel topology.

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by approximately 10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target.

The RNA Helicase DbpA Exhibits a Markedly Different Conformation in the ADP-bound State When Compared with the ATP- or RNA-Bound States

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

Thermodynamics of aminoglycoside and acyl-coenzyme A binding to the Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase.

Kinetic and mechanistic studies on the chromosomally encoded aminoglycoside 6'-N-acetyltransferase, AAC(6')-Iy, of Salmonella enterica that confers resistance toward aminoglycosides have been previously reported [Magnet et al. (2001) Biochemistry 40, 3700-3709]. In the present study, equilibrium binding and the thermodynamic parameters of binding of aminoglycosides and acyl-coenzyme A derivatives to AAC(6')-Iy and of two mutants, C109A and the C109A/C70A double mutant, have been studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Association constants for different aminoglycosides varied greatly (4 x 10(4)-150 x 10(4)) while the association constants of several acyl-coenzyme A derivatives were similar (3.2 x 10(4)-4.5 x 10(4)). The association constants and van't Hoff enthalpy changes derived from intrinsic protein fluorescence changes were in agreement with independently measured values from isothermal titration calorimetry studies. Binding of both aminoglycosides and acyl-coenzyme A derivatives is strongly enthalpically driven and revealed opposing negative entropy changes, resulting in enthalpy-entropy compensation. The acetyltransferase exhibited a temperature-dependent binding of tobramycin with a negative heat capacity value of 410 cal mol(-1) K(-1). Isothermal titration studies of acetyl-coenzyme A and tobramycin binding to mutant forms of the enzyme indicated that completely conserved C109 does not play any direct role in the binding of either of the substrates, while C70 is directly involved in aminoglycoside binding. These results are discussed and compared with previous steady-state kinetic studies of the enzyme.