Biomedical subjects

Stefan Franzen

Publications and source records attributed to Stefan Franzen.

At least 19 recordsLinked to original sources

PubMed2006

Resonance Raman study of ferric heme adducts of dehaloperoxidase from Amphitrite ornata.

The study of axial ligation by anionic ligands to ferric heme iron by resonance Raman spectroscopy provides a basis for comparison of the intrinsic electron donor ability of the proximal histidine in horse heart myoglobin (HHMb), dehaloperoxidase (DHP), and horseradish peroxidase (HRP). DHP is a dimeric hemoglobin (Hb) originally isolated from the terebellid polychaete Amphitrite ornata. The monomers are structurally related to Mb and yet DHP has a peroxidase function. The core size marker modes, v2 and v3, were observed using Soret excitation, and DHP-X was compared to HHMb-X for the ligand series X = F, Cl, Br, SCN, OH, N3, and CN. Special attention was paid to the hydroxide adduct, which is also formed during the catalytic cycle of peroxidases. The Fe-OH stretching frequency was observed and confirmed by deuteration and is higher in DHP than in HHMb. The population of high-spin states of the heme iron in DHP was determined to be intermediate between HHMb and HRP. The data provide the first direct measurement of the effect of axial ligation on the heme iron in DHP. The Raman data support a modified charge relay in DHP, in which a strongly hydrogen-bonded backbone carbonyl (>C=O) polarizes the proximal histidine. The charge relay mechanism by backbone carbonyl >C=O-His-Fe is the analogue of the Asp-His-Fe of peroxidases and Glu-His-Fe of flavohemoglobins.

Animals↗

PubMed2006

The pH dependence of the activity of dehaloperoxidase from Amphitrite ornata.

Dehaloperoxidase (DHP) from the terebellid polychaete, Amphitrite ornata, is the first hemoglobin that has peroxidase activity as part of its native function. The substrate 2,4,6-tribromophenol (TBP) is oxidatively debrominated by DHP to form 2,6-dibromoquinone (DBQ) in a two-electron process. There is a well-defined internal binding site for TBP above the heme, a feature not observed in other hemoglobins or peroxidases. A study of the pH dependence of the activity of DHP reveals a substantial difference in mechanism. From direct observation of the Soret band of the heme it is shown that the pKa for heme activation in protein DHP is 6.5. Below this pH the heme absorbance decreases in the presence of H2O2 with or without addition of substrate. The low pH data are consistent with significant heme degradation. Above pH 6.5 addition of H2O2 causes the heme to shift rapidly to a compound II spectrum and then slowly to an unidentified intermediate with an absorbance of 410 nm. However, the pKa of the substrate TBP is 6.8 and the greatest enzyme activity is observed above the pKa of TBP under conditions where the substrate is a phenolate anion (TPBO-). Although the mechanisms may differ, the data show that both neutral TBP and anionic TPBO- are converted to the quinone product. The mechanistic implications of the pH dependence are discussed by comparison other known peroxidases, which oxidize substrates at the heme edge.

Animals↗

PubMed2006

Proximal cavity, distal histidine, and substrate hydrogen-bonding mutations modulate the activity of Amphitrite ornata dehaloperoxidase.

Dehaloperoxidase (DHP) from Amphitrite ornata is the first globin that has peroxidase activity that approaches that of heme peroxidases. The substrates 2,4,6-tribromophenol (TBP) and 2,4,6-trichlorophenol are oxidatively dehalogenated by DHP to form 2,6-dibromo-1,4-benzoquinone and 2,6-dichloro-1,4-benzoquinone, respectively. There is a well-defined internal substrate-binding site above the heme, a feature not observed in other globins or peroxidases. Given that other known heme peroxidases act on the substrate at the heme edge there is great interest in understanding the possible modes of substrate binding in DHP. Stopped-flow studies (Belyea, J., Gilvey, L. B., Davis, M. F., Godek, M., Sit, T. L., Lommel, S. A., and Franzen, S. (2005) Biochemistry 44, 15637-15644) show that substrate binding must precede the addition of H2O2. This observation suggests that the mechanism of DHP relies on H2O2 activation steps unlike those of other known peroxidases. In this study, the roles of the distal histidine (H55) and proximal histidine (H89) were probed by the creation of site-specific mutations H55R, H55V, H55V/V59H, and H89G. Of these mutants, only H55R shows significant enzymatic activity. H55R is 1 order of magnitude less active than wild-type DHP and has comparable activity to sperm whale myoglobin. The role of tyrosine 38 (Y38), which hydrogen bonds to the hydroxyl group of the substrate, was probed by the mutation Y38F. Surprisingly, abolishing this hydrogen bond increases the activity of the enzyme for the substrate TBP. However, it may open a pathway for the escape of the one-electron product, the phenoxy radical leading to polymeric products.

Animals↗

PubMed2006

Hydrophobic distal pocket affects NO-heme geminate recombination dynamics in dehaloperoxidase and H64V myoglobin.

The recombination dynamics of NO with dehaloperoxidase (DHP) from Amphitrite ornata following photolysis were measured by femtosecond time-resolved absorption spectroscopy. Singular value decomposition (SVD) analysis reveals two important basis spectra. The first SVD basis spectrum reports on the population of photolyzed NO molecules and has the appearance of the equilibrium difference spectrum between the deoxy and NO forms of DHP. The first basis time course has two kinetic components with time constants of tau(11) approximately 9 ps and tau(12) approximately 50 ps that correspond to geminate recombination. The fast geminate process tau(11) arises from a contact pair with the heme iron in a bound state with S = 3/2 spin. The slow geminate process tau(12) corresponds to the recombination from a more remote docking site >3 A from the heme iron with the greater barrier corresponding to a S = 5/2 spin state. The second SVD basis spectrum represents a time-dependent Soret band shift indicative of heme photophysical processes and protein relaxation with time constants of tau(21) approximately 3 ps and tau(22) approximately 17 ps, respectively. A comparison between the more rapid rate constant of the slow geminate phase in DHP-NO and horse heart myoglobin (HHMbNO) or sperm whale myoglobin (SWMbNO) suggests that protein interactions with photolyzed NO are weaker in DHP than in the wild-type MbNOs, consistent with the hydrophobic distal pocket of DHP. The slower protein relaxation rate tau(22) in DHP-NO relative to HHMbNO implies less effective trapping in the docking site of the distal pocket and is consistent with a greater yield for the fast geminate process. The trends observed for DHP-NO also hold for the H64V mutant of SWMb (H64V MbNO), consistent with a more hydrophobic distal pocket for that protein as well. We examine the influence of solution viscosity on NO recombination by varying the glycerol content in the range from 0% to 90% (v/v). The dominant effect of increasing viscosity is the increase of the rate of the slow geminate process, tau(12), coupled with a population decrease of the slow geminate component. Both phenomena are similar to the effect of viscosity on wild-type Mb due to slowing of protein relaxation resulting from an increased solution viscosity and protein surface dehydration.

Amino Acid Substitution↗

PubMed2006

Spectroscopic study of substrate binding to the carbonmonoxy form of dehaloperoxidase from Amphitrite ornata.

Dehaloperoxidase (DHP) is a globular heme enzyme found in the marine worm Amphitrite ornata that can catalyze the dehalogenation of halophenols to the corresponding quinones by using hydrogen peroxide as a cosubstrate. Its three-dimensional fold is surprisingly similar to that of the oxygen storage protein myoglobin (Mb). A key structural feature common to both DHP and Mb is the existence of multiple conformations of the distal histidine. In DHP, the conformational flexibility may be involved in promotion of substrate and cosubstrate entry and exit. Here we have explored the dynamics of substrate binding in DHP using Fourier transform infrared spectroscopy and flash photolysis. A number of discrete conformations at the active site were identified from the appearance of multiple CO absorbance bands in the infrared region of the spectrum. Upon photolysis at cryogenic temperatures, the CO molecules are trapped at docking sites within the protein matrix, as inferred from the appearance of several photoproduct bands characteristic of each site. Substrate binding stabilizes the protein by approximately 20 kJ/mol. The low yield of substrate-bound DHP at ambient temperature points toward a steric inhibition of substrate binding by carbon monoxide.

Animals↗

PubMed2006

Testing bridge-mediated differences in dinuclear valence tautomeric behavior.

Two structurally characterized dinuclear valence tautomers are described. Cobalt ions are bridged by p- and m-phenylene units connected to 2,2'-bipyridines. X-ray crystal structures show that the molecules are in the [(Co(III))(Co(III))] forms at ca. 125 K, while spectroscopic studies show that both molecules can achieve the [(Co(II))(Co(II))] form above 400 K and confirm the [(Co(III))(Co(III))] form below 10 K. Magnetic susceptibility studies are also included. Our results highlight the necessity of studying both crystalline and amorphous samples to distinguish the effects of intrinsic electronic structure and intermolecular forces on valence tautomeric behavior.

Journal Article↗

PubMed2006

Gold and silica-coated gold nanoparticles as thermographic labels for DNA detection.

The infrared emissivity of Au and silica-coated Au nanoparticles (Au NPs) deposited on indium tin oxide substrates was investigated. NPs were irradiated with laser light at a frequency close to the Au plasmon resonance band, and the blackbody radiation emitted as a result was monitored with an IR camera equipped with an InAs array detector. The differences in temperature before and after laser irradiation were recorded (T-jumps) and were found to be directly proportional to the number of particles present on the slide and to the laser power used in the experiment. Coating Au NPs with silica increased the measured T-jumps 2-5 times, depending on the thickness of the silica shell. This was in agreement with the observation that silica has a much higher IR emissivity than Au. Both Au and silica-coated Au NPs were then tested as labels for thermographic DNA detection. Target DNA concentrations as low as 100 pM were recorded when Au NPs were used as labels and as low as 10 pM when silica-coated Au NPs were used.

DNA↗

PubMed2006

Controlled encapsidation of gold nanoparticles by a viral protein shell.

Icosahedral virus capsids demonstrate a high degree of selectivity in packaging cognate nucleic acid components during assembly. This packaging specificity, when integrated as part of a nanotechnological protocol, has the potential to encapsidate a wide array of foreign materials for delivery of therapeutics or biosensors into target cells. Red clover necrotic mosaic virus (RCNMV) exclusively packages two genomic ssRNAs initiated by a specific protein:RNA interaction between the RCNMV coat protein (CP) and the viral RNA origin of assembly (OAS) element. In the present work, an oligonucleotide mimic of the RCNMV OAS sequences is attached to Au nanoparticles as a recognition signal to initiate the virion-like assembly by RCNMV CP. Covalent linkage of the OAS to Au functions as a trigger for specific encapsidation and demonstrates that foreign cargo can be packaged into RCNMV virions.

Capsid Proteins↗

PubMed2006

Role of heme iron coordination and protein structure in the dynamics and geminate rebinding of nitric oxide to the H93G myoglobin mutant: implications for nitric oxide sensors.

The influence of the heme iron coordination on nitric oxide binding dynamics was investigated for the myoglobin mutant H93G (H93G-Mb) by picosecond absorption and resonance Raman time-resolved spectroscopies. In the H93G-Mb, the glycine replacing the proximal histidine does not interact with the heme iron so that exogenous substituents like imidazole may coordinate to the iron at the proximal position. Nitrosylation of H93G-Mb leads to either 6- or 5-coordinate species depending on the imidazole concentration. At high concentrations, (imidazole)-(NO)-6-coordinate heme is formed, and the photoinduced rebinding kinetics reveal two exponential picosecond phases ( approximately 10 and approximately 100 ps) similar to those of wild type myoglobin. At low concentrations, imidazole is displaced by the trans effect leading to a (NO)-5-coordinate heme, becoming 4-coordinate immediately after photolysis as revealed from the transient Raman spectrum. In this case, NO rebinding kinetics remain bi-exponential with no change in time constant of the fast component whose amplitude increases with respect to the 6-coordinate species. Bi-exponential NO geminate rebinding in 5-coordinate H93G-Mb is in contrast with the single-exponential process reported for nitrosylated soluble guanylate cyclase (Negrerie, M., Bouzhir, L., Martin, J. L., and Liebl, U. (2001) J. Biol. Chem. 276, 46815-46821). Thus, our data show that the iron coordination state or the heme iron out-of-plane motion are not at the origin of the bi-exponential kinetics, which depends upon the protein structure, and that the 4-coordinate state favors the fast phase of NO geminate rebinding. Consequently, the heme coordination state together with the energy barriers provided by the protein structure control the dynamics and affinity for NO-binding enzymes.

Animals↗

PubMed2005

Enzyme function of the globin dehaloperoxidase from Amphitrite ornata is activated by substrate binding.

Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.

Animals↗

PubMed2005

Effect of modulating unfolded state structure on the folding kinetics of the villin headpiece subdomain.

Equilibrium Fourier transform infrared (FTIR) and temperature-jump (T-jump) IR spectroscopic techniques were used to study the thermodynamics and kinetics of the unfolding and folding of the villin headpiece helical subdomain (HP36), a small three-helix protein. A double phenylalanine mutant (HP36 F47L, F51L) that destabilizes the hydrophobic core of this protein also was studied. The double mutant is less stable than wild type (WT) and has been shown to contain less residual secondary structure and tertiary contacts in its unfolded state. The relaxation kinetics after a T-jump perturbation were studied for both HP36 and HP36 F47L, F51L. Both proteins exhibited biphasic relaxation kinetics in response to a T-jump. The folding times for the WT (3.23 micros at 60.2 degrees C) and double phenylalanine mutant (3.01 micros at 49.9 degrees C) at the approximate midpoints of their thermal unfolding transitions were found to be similar. The folding time for the WT was determined to be 3.34 mus at 49.9 degrees C, similar to the folding time of the double phenylalanine mutant at that temperature. The double phenylalanine mutant, however, unfolds faster with an unfolding time of 3.01 micros compared with 6.97 micros for the WT at 49.9 degrees C.

Kinetics↗

PubMed2005

Probing BSA binding to citrate-coated gold nanoparticles and surfaces.

The interaction of bovine serum albumin (BSA) with gold colloids and surfaces was studied using zeta-potential and quartz crystal microbalance (QCM) measurements, respectively, to determine the surface charge and coverage. The combination of these two measurements suggests that BSA binding to gold nanoparticles and gold surfaces occurs by an electrostatic mechanism when citrate is present. The binding of BSA to bare gold is nearly two times greater than the binding of BSA to a citrate-coated gold surface, suggesting that protein spreading (denaturation) on the surface may occur followed by secondary protein binding. On the other hand, binding to citrate-coated gold surfaces can be fit to a Langmuir isotherm model to obtain a maximum surface coverage of (3.7 +/- 0.2) x 10(12) molecules/cm(2) and a binding constant of 1.0 +/- 0.3 microM(-1). The zeta-potential measurements show that the stabilization of colloids by BSA has a significant contribution from a steric mechanism because the colloids are stable, even at their isoelectric point (pI approximately 4.6). To be consistent with the observed phenomena, the electrostatic interactions between BSA and citrate must consist of salt-bridges, for example, of the carboxylate-ammonium type, between the citrate and the lysine on the protein surface. The data support the role of strong electrostatic binding but do not exclude contributions from steric or hydrophobic interactions with the surface adlayer.

Journal Article↗

PubMed2005

Hairpin folding dynamics: the cold-denatured state is predisposed for rapid refolding.

Cold denaturation is a general phenomenon in globular proteins, and the associated cold-denatured states of proteins have important fundamental and practical significance. Here, we have characterized the cold-denatured state of a beta-hairpin forming peptide, MrH3a, in 8% hexafluoro-2-propanol (HFIP) and the dynamics of its refolding following a laser-induced T-jump. Beta-hairpins constitute an important class of protein structural elements, yet their folding mechanisms are not fully understood. Characterization of MrH3a using NMR, CD, and IR spectroscopies reveals residual structure in the cold-denatured state, in contrast with the highly disordered heat-denatured state. The residual structure in the cold-denatured state comprises relatively compact and solvent protected conformations. Furthermore, we find a substantial acceleration in the rate of folding from the cold-denatured state compared to that of the heat-denatured state. In addition, the cold-denatured state is not populated in 20% HFIP; folding occurs only from the fully unfolded state and is significantly slower. We interpret the acceleration of the folding rate of MrH3a in 8% HFIP as a direct consequence of the collapsed conformations of the cold-denatured state. Finally, there may be some reduction of the loop search cost when starting from the cold-denatured state, since this state may have some of the stabilizing cross-strand interactions already formed.

Circular Dichroism↗

PubMed2005

Supramolecular control of valence-tautomeric equilibrium on nanometer-scale gold clusters.

This communication describes the preparation and characterization of a valence tautomer complex covalently attached to gold nanoparticles. Variable-temperature IR spectroscopy is used to determine the equilibrium thermodynamic parameters for the valence tautomerization. These results are compared with a nonsurface combined valence tautomer complex. The results show that surface confinement attenuates both DeltaH degrees and DeltaS degrees . We attribute these changes to a reduced accessible molecular surface area.

Journal Article↗

PubMed2005

Solvatochromism of a novel betaine dye derived from purine.

A novel solvatochromic betaine dye has been synthesized from xanthosine and characterized spectroscopically by UV-vis in a broad range of solvents. The dye 9-(2',3',5'-tri-O-acetyl-beta-d-ribofuranosyl)-2-(pyridinium-1-yl)-9H-purin-6-olate, 1a, exhibits solvent-induced spectral band shifts that are (2)/(3) as large as that of the betaine known as Reichardt's dye, which forms the basis of the E(T)(30) solvent polarity scale. Moreover, the dye 1a is a ribonucleoside and hence has the potential application as a polarity probe for application in RNA oligonucleotides. The isomeric dye 6-(pyridinium-1)-yl-9H-purin-2-olate, 2a, has also been synthesized and exhibits slightly smaller solvatochromic band shifts. The new betaine dyes have also been studied by comparing the experimental and calculated solvatochromic shifts based on the calculation of the UV/vis absorption spectra, using a combination of methods with density functional theory (DFT). The COSMO continuum dielectric method, an applied electric field term in the Hamiltonian, and time-dependent density functional theory (TD-DFT) methods were used to obtain absorption energies, ground-state dipole moments, and the difference dipole moment between the ground and excited states. The calculations predict a lower energy absorption band of charge-transfer character that is highly solvatochromic, and a higher energy absorption band that has pi-pi character which is not solvatochromic, in agreement with the experimental data. For Reichardt's dye the difference dipole moment between the ground and excited state (Deltamu = mu(e) - mu(g)) was also calculated and compared to experiment: Deltamu(calcd) = -6 D and Deltamu(exptl) = -9 +/- 1 D.(1) The ground-state dipole moment was found to be mu(g)(calcd) = 18 D and mu(g)(exptl) = 14.8 +/- 1.2 D.(1).

Betaine↗

PubMed2005

Detection of adsorption of Ru(II) and Os(II) polypyridyl complexes on gold and silver nanoparticles by single-photon counting emission measurements.

The present study describes a new application of ruthenium(II) tris(bipyridine) (Ru(bpy)3(2+)) and osmium(II) tris(bipyridine) (Os(bpy)3(2+)) as phosphorescent labels for the quantification of surface binding of molecules to gold and silver nanoparticles. The fraction of Ru(bpy)3(2+) and Os(bpy)3(2+) that is in solution can be distinguished from the surface-bound fraction by the relative lifetimes and integrated emission yields as determined by time-correlated single-photon counting (TCSPC) spectroscopy. Complementary steady-state measurements were carried out to confirm surface attachment of the phosphorescent label molecules. Although the emission of solutions of Ru(bpy)3(2+) and Os(bpy)3(2+) is quenched proportional to the concentration of 10 nm Au or 20 nm Ag nanoparticles, the quenching is static and not diffusional quenching observed in Stern-Volmer plots. The results demonstrate that time-resolved spectroscopy provides a rapid method for the measurement of surface binding of labeled molecules on metallic nanoparticles. While steady-state measurements require the preparation of a series of samples with varying quencher concentrations and a reference, the method described herein requires a single sample plus reference. The mechanism for phosphorescence quenching on Au and Ag nanoparticles is discussed in terms of energy and electron transfer theories.

Adsorption↗

PubMed2005

The origin of stark splitting in the initial photoproduct state of MbCO.

Ligand migration and binding in heme proteins have been measured by X-ray diffraction and time-resolved spectroscopy of photoproduct intermediates. In myoglobin (Mb), internal cavities serve as docking sites for carbon monoxide (CO) ligands. In these sites, the CO ligands display characteristic infrared (IR) stretching bands due to interactions with the local electrical field. In the primary docking site, a CO can reside in two opposite orientations, characterized by a doublet of infrared bands, B1 at approximately 2130 and B2 at approximately 2120 cm-1. To assign these bands to the specific orientations, we have reexamined the effects of mutating His64 and Val68 on the infrared stretching bands associated with the B1 and B2 photoproduct states. Wild-type, H64L, V68F, and H64L-V68F MbCO were selected for experimental and theoretical analyses. Fourier transform infrared (FTIR) spectroscopy and density functional theory (DFT) calculations were used to interpret the effects of the electrostatic environment on the B state bands. The imidazole side chain of His64 appears to be the primary cause of the observed Stark splitting. The high-frequency B1 band is assigned to the CO orientation in which the carbon (white atom) is directed toward the heme iron and the Nepsilon-H proton of His64. At low temperatures, CO molecules in the opposite orientational conformer, B2 with the O atom (red) toward His64, first rotate by 180 degrees into the more stable B1 state and then rebind.

Carbon Monoxide↗

PubMed2005

Assembly and characterization of biomolecule-gold nanoparticle conjugates and their use in intracellular imaging.

In this chapter, we outline protocols for assembling and characterizing peptide-gold nanoparticle conjugates. We describe two strategies for attaching peptides to gold nanoparticles. One involves the covalent coupling of cysteine-terminated peptides directly to a particle surface via a sulfur-gold bond. Alternatively, peptides are coupled to bovine serum albumin (BSA) via a bifunctional molecular crosslinker. We also describe a number of characterization methods for determining the number of crosslinkers per BSA, peptides per BSA, and peptides adsorbed per particle. Finally, we show that the enormous visible light extinction properties of gold nano particles make them excellent imaging agents for tracking the trajectories of peptides inside living cells.

Animals↗