A Euclidean perspective on the unfolding of azurin: spatial correlations

We investigate the stability to structural perturbation of Pseudomonas aeruginosa azurin using a previously developed geometric model. Our analysis considers Ru(2,2′,6′,2^″-terpyridine)(1,10-phenanthroline)(His83)-labelled wild-type azurin and five variants with mutations to Cu-ligating residues. We find that in the early stages of unfolding, the β-strands exhibit the most structural stability. The conserved residues comprising the hydrophobic core are dislocated only after nearly complete unfolding of the β-barrel. Attachment of the Ru-complex at His83 does not destabilize the protein fold, despite causing some degree of structural rearrangement. Replacing the Cys112 and/or Met121 Cu ligands does not affect the conformational integrity of the protein. Notably, these results are in accord with experimental evidence, as well as molecular dynamics simulations of the denaturation of azurin.     

Orientation selective ESEEM studies on the blue oxidases laccase and ascorbate oxidase

The blue oxidases, laccase and ascorbate oxidase, contain three spectroscopically distinct copper binding sites, two of which are EPR detectable in the oxidized Cu(II) state, called type 1 (T1) and type 2 (T2). The three dimensional structure of ascorbate oxidase has recently been determined (Messerschmidt A.et al.: J. Mol. Biol.206, 513 (1989)) while that of laccase has not. We have therefore carried out comparative electron spin echo envelope modulation (ESEEM) measurements on ascorbate oxidase, laccase and laccase in which T1 Cu(II) was substituted with Hg(II) in order to obtain structural information about the copper sites in laccase. The ESEEM results clearly show that there are as many histidines in laccase as in ascorbate oxidase, i.e., at least two at each site. Orientation selective ESEEM experiments showed that in the T1 site in both enzymes the two remote (uncoordinated) nitrogens are magnetically inequivalent and have different hyperfine interactions. Furthermore, the isotropic hyperfine constants of both remote nitrogens in laccase T1 are larger than those in ascorbate oxidase T1. In laccase T2 two remote nitrogens show similar hyperfine couplings and the modulation depth is significantly deeper than in ascorbate oxidase. Finally, it is suggested that the difference between the NQR frequencies of the remote nitrogens in T1 and T2 in both oxidases can be attributed to the alkyl group of the side chain being adjacent to the bound imidazole in T1 and to the remote nitrogen in T2. This is in accordance with the known X-ray structure of ascorbate oxidase.     

Can soaked‐in scavengers protect metalloprotein active sites from reduction during data collection?

One of the first events taking place when a crystal of a metalloprotein is exposed to X‐ray radiation is photoreduction of the metal centres. The oxidation state of a metal cannot always be determined from routine X‐ray diffraction experiments alone, but it may have a crucial impact on the metal's environment and on the analysis of the structural data when considering the functional mechanism of a metalloenzyme. Here, UV–Vis microspectrophotometry is used to test the efficacy of selected scavengers in reducing the undesirable photoreduction of the iron and copper centres in myoglobin and azurin, respectively, and X‐ray crystallography to assess their capacity of mitigating global and specific radiation damage effects. UV–Vis absorption spectra of native crystals, as well as those soaked in 18 different radioprotectants, show dramatic metal reduction occurring in the first 60 s of irradiation with an X‐ray beam from a third‐generation synchrotron source. Among the tested radioprotectants only potassium hexacyanoferrate(III) seems to be capable of partially mitigating the rate of metal photoreduction at the concentrations used, but not to a sufficient extent that would allow a complete data set to be recorded from a fully oxidized crystal. On the other hand, analysis of the X‐ray crystallographic data confirms ascorbate as an efficient protecting agent against radiation damage, other than metal centre reduction, and suggests further testing of HEPES and 2,3‐dichloro‐1,4‐naphtoquinone as potential scavengers.     

Heavy metal coordination chemistry in mercaptides and enzymes studied by TDPAC

Time differential perturbed angular correlation (TDPAC) studies of the coordination chemistry of the heavy metal atoms Cd and Hg via the nuclear quadrupole interaction are presented for the following systems; (i) mercury complexes with mercaptides, polymers with thiol groups, and ferrocenethiols. Mercury has a strong tendency to form linear or almost linear bonds with sulfur ligands. Evidence for 1,3-dithia-2-mercura[3]ferrocenophane formation is presented. (ii)^111mCd-derivatives of the small electron transport proteins azurin, including a his 117gly mutant, and stellacyanin. The titration of the his 117gly mutant of azurin with imidazole was monitored in situ. (iii)^111mCd- and^199mHg-derivatives of the multi-Cu enzymes ascorbate oxidase and laccase. Reconstitution probabilities for Hg-reconstitution will be given as well as information on selective depletion and blocking of Cu-sites.     

Extrinsic fluorescence probe study of human serum albumin using Nile red

Nile red bound to human serum albumin (HSA) shows an order of magnitude increase in the probe's fluorescence intensity. Here, we report on the fluorescence characteristics of the probe-protein complex in Trizma buffer (pH 7.1), urea, guanidine hydrochloride, and AOT/isooctane/buffer reverse micelles using both steady—state and time-resolved fluorescence techniques. With a view to illustrating the use of extrinsic probe fluorescence spectroscopy in protein research, we demonstrate that protein unfolding can be observed through measurements of the probe's time-resolved anisotropy and steady-state fluorescence spectrum. Moreover, this shows that thermal unfolding is fundamentally different from using denaturant, with respect to changes in both the nanosecond diffusional rotation of the probe at intermediate stages and in the denatured protein's structure. Also, the large Stokes shift of Nile red allows the changes in the environment of the probe-protein complex in reverse micelles of varying waterpool size to be easily identified in the steady-state fluorescence. This was not seen in earlier work exploiting the intrinsic tryptophan fluorescence of HSA and further demonstrates the complementary information that extrinsic fluorescence probe studies can offer protein science. We discuss the complex acrylamide quenching characteristics of Nile red bound to HSA in terms of the possibility of at least two binding sites for the probe and the effect of acrylamide on the probe-protein structure at very high quencher concentrations.     

Contributions of polar and apolar groups to the thermodynamic stability of azurin

Summary All denaturational thermodynamic changes (ΔH, ΔS, ΔG) were calculated for the thermal unfolding of azurin fromPseudomonas Aeruginosa. DSC data could not be directly studied in terms of classical thermodynamics, because the thermal unfolding of azurin is an irreversible process. However, a mathematical extrapolation of heat capacity curves to infinite scan rate allowed us to obtain the denaturational enthalpy change and the melting temperature related to the reversible step. The denaturational heat capacity change (ΔC _p ), together with all the relevant contributions of polar and apolar groups to the thermodynamic parameters of azurin were calculated according to an approach taking into account the common features of protein unfolding and dissolution of model compounds. A comparison between the experimental and the expected denaturational thermodynamic values was briefly discussed.     

Role of vibronic coupling and of conformational substate distribution in determining the features of copper-protein EPR spectra

It is stressed that the presence of a metal ion directly bound to a protein can introduce elements of complexity, such as vibronic coupling, which cannot be ruled outa priori. As a typical example, two copper proteins, azurin and plastocyanin, have been considered. Electron paramagnetic resonance spectra of the two proteins, recorded in the range 4–200 K and with different cooling rates, support the hypothesis of a prominent role played by vibronic interactions as well as by the conformational substate distribution.     

Effect of hydrostatic pressure on the fluorescence of indole derivatives

Effects of hydrostatic pressure on the fluorescence emission of L-tryptophan, N-acetyl-L-trytophanamide and indole were investigated. An increase in pressure ranging from 1 bar to 2.4 kbar results in reversible red-shifts of the emission of the three fluorophores. The pressure-induced redshift amounts to about 170 cm^–1 at 2.4 kbar, and appears related to changes in Stokes shift of the fluorophores caused by pressure effects on the dielectric constant and/or refractive index of the medium. As the pressure range investigated here is the range commonly used in studies of protein subunit association and/or folding, these observations raise the need for caution in interpreting pressure-induced spectral shifts. The significance of these observations to pressure studies of proteins is illustrated by investigation of pressure effects on human Cu,Zn Superoxide dismutase (SOD) and azurin fromPseudomonas aeruginosa. A reversible 170 cm^–1 red-shift of the emission of SOD was observed upon pressurization to 2.4 kbar. This might be interpreted as pressure-induced conformational changes of the protein. However, further studies using SOD that had been fully unfolded by guanidine hydrochloride, and fluorescence anisotropy measurements indicated that the observed red-shift was likely due to a direct effect of pressure on the fluorescence of the single tryptophan residue of SOD. Similar pressure-induced red-shifts were also observed for the buried tryptophan residue of azurin or for azurin that had been previously denatured by guanidine hydrochloride. These observations further suggest that the effective dielectric constant of the protein matrix is affected by pressure similarly to water.     

Azurin-Solvent interaction: an ESR spin labeling investigation

We have investigated by means of electron spin resonance (ESR) spectroscopy using two spin labels, Iodoacetamido-proxyl and 3-Maleimido-proxyl, the dynamics of two different regious around the active site of azurin, a copper containing blue protein. The ESR measurements of spin labeled azurin have been carried out in the 110–300 K temperature range on wet (H₂O, D₂O and ethanol/water mixtures) and lyophilized samples. The behaviours of the outer hyperfine splitting separation, 2A′ _zz , of the ESR spectravs temperature of the lyophilized, and fully hydrated azurin in H₂O and D₂O suggest that the two spin labels are located in regions of the protein surface with different dynamics and polarity. Moreover, all differences in the 2A′ _zz values shown by the spin labeled azurin in normal and heavy water as well as the temperature behaviour disappear when azurin is in ethanol/water mixtures. The results are discussed in terms of a close correlation between the molecular dynamics of the protein fragments to which the two spin labels are bound and the properties of the solvent used.     

Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.     

具有电子推拉结构的多支化分子的光物理特性的研究

利用稳态光谱和飞秒时间分辨荧光亏蚀的技术,研究了不同溶剂中一系列有分子内电荷转移特性的分子的结构与光物理性质的关系,研究体系为三苯胺作为电子给体,2,1,3-苯并噻二唑作为受体的单支分子及其对应的两支和三支分子. 并结合TD-DFT计算进一步解释了实验中所观察到的现象. 三个分子相似的吸收和荧光光谱以及强的溶剂依赖光谱特性表明两支与三支分子激发态与单支分子相似,表明激发态都定域在其中一支上. 激发时多支分子内发生多维电荷转移,然后快速地定域到某一支上发射. 另一方面多支分子相对于单支分子吸收和发射光谱的红     

Protein folding as a stochastic process

In physiological conditions globular protein molecules assume a specific native conformation uniquely determined by its amino acid sequence. Upon environmental changes the protein molecules undergo reversible unfolding (order losing) and folding (order gaining) transitions, which is similar to the first-order phase transition. Pathways of folding have been intensively studied in the hope of deciphering the code that amino acid sequences carry as to the threedimensional structure of proteins. A strongly simplifiedlattice model of proteins has been found to be a powerful theoretical tool to simulate the dynamic process of the folding and unfolding transitions. The results of the simulation indicate the existence of stochastic pathways of folding.     

Nuclear quadrupole interaction of111Cd on type-1 Cu-sites in blue copper proteins

The nuclear quadrupole interaction (NQI) of¹¹¹Cd substituted for Cu(II) on type-1 sites in blue copper proteins is characterized by high values of ₀ in the region of 300 Mrad/s, close to that for the catalytic zinc site in alcohol dehydrogenase. Type-1 Cu has usually two sulfur ligands and two nitrogen ligands and in some cases an oxygen ligand in either a distorted tetrahedral geometry or in a trigonal bipyramidal geometry. The near tetrahedral arrangement together with the ligand sphere containing the same number of sulfur ligands explains the value of ₀ in the blue copper proteins. The present work determined the partial NQI for methionine using the known structure of azurin. This value was then used in the angular overlap model to calculate the NQI for ascorbate oxidase the structure of which is also known and gave good agreement with experiment. NQI data for laccase and stellacyanin the structures of which are unknown, are also given.On leave from Technische Universität München, Germany.     

Atomic dimer shuttling and two-level conductance fluctuations in Nb nanowires

We present high-resolution conductance measurements in niobium nanowires below the superconducting transition temperature. During elongation we find a bistability region manifesting itself as random telegraph noise. Density functional structural optimizations and conductance calculations reproduce and explain the measurements. In particular, the observed bistability is associated with the formation of a niobium dimer between the opposing electrodes, with the dimer shuttling between a symmetric, high conductance, and an asymmetric, low-conductance configurations in the gap.     

Effect of unfolding on the thickness of the hydration layer of a protein

Properties of water in the hydration layer around a protein is intimately correlated with its function. A knowledge of the thickness of the hydration layer is important to understand its role in guiding the folding-unfolding of the protein. We have performed atomistic molecular dynamics simulations of the folded native and a partially unfolded molten globule structure of the villin headpiece subdomain or HP-36 in aqueous solution to estimate the effect of unfolding on the thickness of hydration layer around different segments of the protein. In particular, several dynamic properties of water around different segments of the folded native and the unfolded structure have been calculated by varying the thickness of the hydration layers. It is found that unfolding of a segment of the protein is correlated with the dynamics of water around it, i.e., within the first hydration layer. The effect of unfolding on water properties has been found to diminish when water molecules present beyond the first hydration layer were included in the calculations.      

Fluorescence Analysis of Acridine Orange Adsorbate at the Water/N-Heptane Interface, Bulk and Interface

The amphiphilic nature of 3, 6-bisdimethylaminoacridine (trivial name, acridine orange) can be used to characterize (polar/nonpolar) interfaces. This paper studies the properties of acridine orange and its dodecyl derivative at a solvent interface by internal reflection and tension determinations. In the case of absorbance measurements of crystallized acridine orange, the potenz value of the conventional acid dissociation constant pK _a was found to be 9.6. A clear isosbestic point could be obtained after purification at a unique pH for different concentrations. In contrast, in the absorbance measurements of acridine orange dodecyl, no isosbestic point was observed; this was attributed to the free long dodecyl chain. The overlap in the absorbance spectrum and the normalized fluorescence spectrum was found in many solvents around 500 nm. Neutral species were more likely to transfer into organic phase upon shaking. For fluorescence measurements both steady-state and time-resolved spectra were investigated. Results showed that at the interface a bathochromic shift happened to the maxima. This means that chromophore species aggregated at the interface as the dimer or trimer or the macro oligomer. Fluorescence polarization in bulk was calculated and found to be ca. 0.4. For interfacial measurements a dynamic volume method was applied to obtain the interfacial tension. An extrapolated Gibbs plot was obtained. Increasing bulk concentration increased the concentration at the interface to a finite value representing saturation.     

Complexation of insecticide chlorantraniliprole with human serum albumin: Biophysical aspects

Chlorantraniliprole is a novel insecticide belonging to the diamide class of selective ryanodine receptor agonists. A biophysical study on the binding interaction of a novel diamide insecticide, chlorantraniliprole, with staple in vivo transporter, human serum albumin (HSA) has been investigated utilizing a combination of steady-state and time-resolved fluorescence, circular dichroism (CD), and molecular modeling methods. The interaction of chlorantraniliprole with HSA gives rise to fluorescence quenching through static mechanism, this corroborates the fluorescence lifetime outcomes that the ground state complex formation and the predominant forces in the HSA-chlorantraniliprole conjugate are van der Waals forces and hydrogen bonds, as derived from thermodynamic analysis. The definite binding site of chlorantraniliprole in HSA has been identified from the denaturation of protein, competitive ligand binding, and molecular modeling, subdomain IIIA (Sudlow's site II) was designated to possess high-affinity binding site for chlorantraniliprole. Moreover, using synchronous fluorescence, CD, and three-dimensional fluorescence we testified some degree of HSA structure unfolding upon chlorantraniliprole binding.     

Unfolding Features of Bovine Testicular Hyaluronidase Studied by Fluorescence Spectroscopy and Fourier Transformed Infrared Spectroscopy

Chemical unfolding of bovine testicular hyaluronidase (HAase) has been studied by fluorescence spectroscopy and Fourier transformed infrared spectroscopy (FTIR). Thermodynamic parameters were determined for unfolding HAase from changes in the intrinsic fluorescence emission intensity and the formations of several possible unfolding intermediates have been identified. This was further confirmed by representation of fluorescence data in terms of ‘phase diagram’. The secondary structures of HAase have been assigned and semiquantitatively estimated from the FTIR. The occurrence of conformational change during chemical unfolding as judged by fluorescence and FTIR spectroscopy indicated that the unfolding of HAase may not follow the typical two-state model.     

Sensitive Spectroscopic Detection of Large and Denatured Protein Aggregates in Solution by Use of the Fluorescent Dye Nile Red

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein β-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of β-galactosidase below and above the protein’s unfolding temperature of 57.4°C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with β-galactosidase aggregates led to a shift of the emission maximum (λ _max) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated β-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native β-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with β-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.     

Application of Atomic Fluorescence Spectrometry to the Determination of Sulfide Ion in Sewage Waters

Atomic fluorescence spectrometry and direct flame photometry are evaluated as alternative methods for the determination of inorganic sulfide in municipal sewage. The sulfide is volatilized as H₂S from the sewage influent or effluent sample, which has been stabilized by a 0.05 M sodium ascorbate buffer. Both flame spectrometric methods are more sensitive than the standard colorimetric method, but quantification is easier for the atomic fluorescence method. The latter technique has a wide linear dynamic range and is unaffected by a gradual increase in the residual H₂S level in the system. The atomic fluorescence signal is also unaffected by the presence of volatile hydrocarbons in the sewage. The results obtained with the fluorescence method agree well with those obtained with the standard method, while the flame photometric results are low. Studies on the effect of metal ions on the fluorescence signal indicate that the method determines “total sulfide”.