635 research outputs found
Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR
No full textSome organisms can survive complete dehydration and high temperatures by
adopting an anhydrobiotic state in which the intracellular medium contains
large amounts of disaccharides, particularly trehalose and sucrose. Trehalose
is most effective also in protecting isolated in vitro biostructures. In an
attempt to clarify the molecular mechanisms of disaccharide bioprotection, we
compared the structure and dynamics of sucrose and trehalose matrices at
different hydration levels by means of high-field W-band EPR and FTIR
spectroscopy. The hydration state of the samples was characterized by FTIR
spectroscopy and the structural organization was probed by EPR using a
nitroxide radical dissolved in the respective matrices. Analysis of the EPR
spectra showed that the structure and dynamics of the dehydrated matrices as
well as their evolution upon re-hydration differ substantially between
trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms
of distribution of the residual water and spin-probe molecules. In contrast,
dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose
polycrystalline clusters and several bulk water domains. The amorphous form
was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a
structural homogenization of the sucrose matrix, whilst in the trehalose
matrix several domains develop differing in the local water/radical content
and radical mobility. The molecular model of the matrices provides an
explanation for the different protein–matrix dynamical coupling observed in
dried ternary sucrose and trehalose matrices, and accounts for the superior
efficacy of trehalose as a bioprotectant. Furthermore, for bacterial
photosynthetic reaction centers it is shown that at low water content the
protein–matrix coupling is modulated by the sugar/protein molar ratio in
sucrose matrices only. This effect is suggested to be related to the
preference for sucrose, rather than trehalose, as a bioprotective disaccharide
in some anhydrobiotic organisms
Soft Dynamic Confinement of Membrane Proteins by Dehydrated Trehalose Matrices: High-Field EPR and Fast-Laser Studies
No full textIn memory of the 85th birthday of Yakov S. Lebedev (Moscow), who died in 1996, we start this Review on soft-glass matrix effects in donor–acceptor complexes with an appreciation of his pioneering work on high-field EPR spectroscopy on tribochemically generated donor–acceptor complexes. The mechanochemical activation of polycrystalline mixtures of porphyrins (and other donors) and quinone acceptors was found to produce large concentrations of triplet donor molecules and donor–acceptor radical pairs with unusual stability. The Review is continued with reporting on W-band high-field EPR and fast-laser studies on disaccharide matrix effects on structure and dynamics of donor–acceptor protein complexes related to photosynthesis, including the non-oxygenic bacterial reaction center (RC) and the oxygenic RCs Photosystem I (PS I) and Photosystem II (PS II, preliminary results). Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, in particular trehalose and sucrose. Trehalose is most effective in protecting biostructures, both in vivo and in vitro. To clarify the molecular mechanisms of disaccharide bioprotection, structure and dynamics of sucrose and trehalose matrices at different controlled hydration levels were probed by perdeuterated nitroxide spin labels and native cofactor intermediates in their charge-separated states. Trehalose forms a homogeneous amorphous phase in which the hosted molecules are uniformly distributed. Notably, their rotational mobility at room temperature is dramatically impaired by the trehalose H-bonding network confinement to an extent that in normal protein–matrix systems is only observed at low temperatures around 150 K. From the experimental results, formation of an extended H-bonding network of trehalose with protein molecules is inferred, involving both bulk and local water molecules. The H-bond network extends homogeneously over the whole matrix integrating and immobilizing the hosted protein. Taken together, these observations suggest that in photosystems, such as bacterial RCs and PS I complexes, of different size and complexity regarding subunit composition and oligomeric organization, the molecular configuration of the cofactors involved in the primary processes of charge separation is not significantly distorted by incorporation into trehalose glass, even under extensive dehydration. By means of pulsed W-band high-field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR, in conjunction with using isotope labeled water (D2O and H217O), the biologically important issue of sensing and quantification of local water in proteins is addressed. The bacterial RC embedded into the trehalose glass matrix is used as model system. The two native radical cofactor ions of the primary electron donor and acceptor as well as an artificial nitroxide spin label site-specifically attached to the protein surface are studied in the experiments. The three paramagnetic reporter groups probe distinctly different local environments. They sense water molecules via their magnetic hyperfine and quadrupole interactions with either deuterons or 17O nuclei. It is shown that by using oxygen-17 labeled water, quantitative conclusions can be drawn differentiating between local and bulk water. It is concluded that dry trehalose operates as anhydrobiotic protein stabilizer by means of selective changes in the first solvation shell of the protein upon trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding network. Such changes usually have an impact on the global function of a biological system. Finally, preliminary results of optical and W-band EPR experiments on the extremolytes ectoine and its derivative hydroxyectoine are reported; these compounds appear to share several stress-protecting properties with trehalose in terms of stabilizing protein matrices. For instance, they display remarkable stabilizing capabilities towards sensitive proteins and enzymes with respect to freeze-thawing, heat-treatment, and freeze-drying procedures. Moreover, hydroxyectoine is a good glass-forming compound and exhibits a remarkable bioprotective effect against desiccation and heat denaturation of functional protein complexes
The Magic of Disaccharide Glass Matrices for Protein Function as Decoded by High-Field EPR and FTIR Spectroscopy
No full textThe structural and dynamical interaction of proteins with their microenvironment in disordered matrices plays a decisive role for their function; EPR spectroscopy is a powerful tool for shading light onto the molecular mechanisms of this protein-matrix interplay. To clarify the molecular mechanisms of disaccharide bioprotection, we studied the structure and dynamics of spin-labeled systems and photosynthetic reaction centers (RCs) in sucrose and trehalose matrices at different hydration levels by means of cw and pulse high-field 95 GHz (W-band) EPR as well as by FTIR. In this minireview, we summarize and discuss EPR and FTIR experiments showing that the anhydrobiotic state of the RC-trehalose system (1) is not the result of matrix-induced changes of the local structure of the charge-separated radical-pair cofactors, and , and (2) is not the result of changes of local dynamics and local hydrogen bonding of Q(A) in its binding pocket. Rather, the extreme impairment of RC dynamics caused by incorporation into the dehydrated trehalose matrix, which also protects it against thermal denaturation, originates in the high rigidity, already at room temperature, of the dry trehalose glass matrix coating the RC protein surface. This surface hydrogen-bonding scaffold shifts the correlation time of thermal conformational fluctuations into the non-biological time domain. Another intriguing aspect of disaccharide bioprotection is the superior efficiency of trehalose versus sucrose matrices in stabilizing the anhydrobiotic state of proteins. To clarify the molecular basis of this specificity, glassy trehalose-water and sucrose-water binary systems, incorporating a nitroxide radical as spin probe, have been studied by high-field W-band EPR spectroscopy at different water contents. Analysis of the EPR spectra revealed a different structural and dynamical organization in the sucrose and trehalose matrix, only the trehalose being homogeneous in terms of residual water and nitroxide distribution
Influence of the axial ligands on the spectral properties of P700 of photosystem I: A study of site-directed mutants
No full textTwo histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+.)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Kass, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+.)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+.), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656)
The radical cation of bacteriochlorophyll b. A liquid-phase endor and triple resonance study
No full textThe previous termradical cationnext term of bacterioehlorophyll b (BChl b) is investigated by ENDOR and TRIPLE resonance in liquid solution. The experimental hyperfine coupling constants, ten proton and three nitrogen couplings, are compared with the predictions from advanced molecular-orbital calculations (RHF INDO/SP). The detailed picture obtained of the spin density distribution is a prerequisite for the investigation of the primary electron donor previous termradical cationnext term in BChl b containing photosynthetic bacteria
Modulation of apoptosis by adenosine in the central nervous system: a possible role for the A3 receptor. Pathophysiological significance and therapeutic implications for neurodegenerative disorders
No full textUntersuchung der Struktur und Dynamik von T4 Lysozym auf planaren Oberflächen mittels ESR-Spektroskopie
No full textEs ist eine allgemein akzeptierte Tatsache, dass der Kontakt von Proteinen mit synthetischen Materialien üblicherweise zur Proteinadsorption an der Materialoberfläche führt. Über den stattfindenden Prozess, insbesondere das Zusammenspiel zwischen Protein-Oberflächen-Wechselwirkungen und konformellen Änderungen der adsorbierten Proteine ist jedoch bisher nur wenig bekannt. In dieser Arbeit wird die ortsgerichtete Spinmarkierungstechnik (SDSL) auf die Strukturuntersuchung adsorbierter Proteine ausgeweitet. Diese nutzt das spezifische Einbringen einer spinmarkierte Seitenkette an gewünschte Positionen der Primärstruktur zur Analyse der Struktur und Dynamik diamagnetischer Proteine mittels der Elektronenspinresonanz(ESR)-Spektroskopie. Das globuläre Protein T4 Lysozym (T4L) wurde auf planare Modelloberflächen adsorbiert und strukturelle Änderungen in Abhängigkeit der physikalischen und chemischen Eigenschaften der Oberfläche verfolgt. Die spezifische Anbindung von T4L auf quarzgestützten zwitterionische Lipiddoppelschichten führt nur zu geringfügigen strukturellen Veränderungen des Proteins. Allerdings bildet sich eine makroskopisch geordnete Proteinschicht aus. Die Vorzugsrichtung der Proteine auf der Oberfläche kann durch Analyse der winkelabhängigen ESR-Spektren bestimmt werden. Die Wechselwirkung negativ geladener Oberflächen mit dem positiv geladenen T4L führt zu drastischeren Störungen der Proteinstruktur. Hierbei wird die Reaktion des Proteins auf den Kontakt mit einer fluiden quarzgestützten Lipiddoppelschicht, die das negativ geladenen Lipid Phosphatidylserin enthält, mit derer bei Adsorption auf einer ebenfalls negativ geladenen, jedoch rigiden Quarzoberfläche verglichen. Dass der Adsorptionsprozess auch das Substrat selbst beeinflussen kann, wird durch die Beobachtung einer Phasentrennung bei Proteinadsorption des Lipidgemischs aufgezeigt, das negativ geladene Lipide enthält.Although it is commonly accepted that the exposition of proteins to man-made materials typically results in protein adsorption on the material surface, little is known about the interplay between the protein-surface interactions involved and the resulting conformational changes of the adsorbing protein. In this study the site-directed spin labeling (SDSL) approach has been extended to the investigation of proteins adsorbed to planar surfaces. The method involves the selective introduction of an artificial spin-labeled side-chain to a predefined residue of the amino acid sequence and allows the determination of the structure and dynamics of proteins by analysis of the electron paramagnetic resonance (EPR) spectra. The globular protein T4 Lysozyme (T4L) has been adsorbed to planar model surfaces to study the correlation between conformational changes of the protein and the physical and chemical properties of the surfaces. Tethering T4L to a planar quartz-supported zwitterionic lipid bilayer shows only minor changes in the structure of the protein. Furthermore, a macroscopic order of the adsorbed protein layer is proven by angular-dependent EPR spectra which allow the determination of the protein orientation. Offering surfaces that are net negatively charged to the highly positively charged T4L leads to the observation of more drastic conformational changes. Here, the conformation of T4L adsorbing to a fluid quartz-supported lipid bilayer containing negatively charged lipids is compared to the structure of T4L adsorbed to the negatively charged but rigid quartz surface. The adsorption process may also influence the substrate itself. This can be shown by the phase separation of the negatively charged lipid bilayer upon protein adsorption
Light and Temperature Control of the Spin State of Bis(<i>p</i>‑methoxyphenyl)carbene: A Magnetically Bistable Carbene
No full textBis(<i>p</i>-methoxyphenyl)carbene
is the first carbene
that at cryogenic temperatures can be isolated in both its lowest
energy singlet and triplet states. At 3 K, both states coexist indefinitely
under these conditions. The carbene is investigated in argon matrices
by IR, UV–vis, and X-band EPR spectroscopy and in MTHF glasses
by W-band EPR and Q-band ENDOR spectroscopy. UV (365 nm) irradiation
of the system results in formation of predominantly the triplet carbene,
whereas visible (450 nm) light shifts the photostationary equilibrium
toward the singlet state. Upon annealing at higher temperatures (>10
K), the triplet is converted to the singlet; however, cooling back
to 3 K does not restore the triplet. Therefore, depending on matrix
temperature and irradiation conditions, matrices containing predominantly
the triplet or singlet carbene can be generated. Controlling the magnetic
and chemical properties of carbenes by using light of different wavelengths
might be of general interest for applications such as information
storage and radical-initiated polymerization processes
water exchange in bacterial photosynthetic reaction centers embedded in a trehalose glass studied using multiresonance EPR
No full textUsing isotope labeled water (D2O and H217O) and pulsed W-band (94 GHz) high-
field multiresonance EPR spectroscopies, such as ELDOR-detected NMR and ENDOR,
the biologically important question of detection and quantification of local
water in proteins is addressed. A bacterial reaction center (bRC) from
Rhodobacter sphaeroides R26 embedded into a trehalose glass matrix is used as
a model system. The bRC hosts the two native radical cofactor ions Image
ID:c7cp03942e-t1.gif (primary electron donor) and Image ID:c7cp03942e-t2.gif
(primary electron acceptor) as well as an artificial nitroxide spin label
site-specifically attached to the surface of the H-protein domain. The three
paramagnetic reporter groups have distinctly different local environments.
They serve as local probes to detect water molecules via magnetic interactions
(electron–nuclear hyperfine and quadrupole) with either deuterons or 17O
nuclei. bRCs were equilibrated in an atmosphere of different relative
humidities allowing us to control precisely the hydration levels of the
protein. We show that by using oxygen-17 labeled water quantitative
conclusions can be made in contrast to using D2O which suffers from
proton–deuterium exchange processes in the protein. From the experiments we
also conclude that dry trehalose operates as an anhydrobiotic protein
stabilizer in line with the “anchorage hypothesis” of bio-protection. It
predicts selective changes in the first solvation shell of the protein upon
trehalose–matrix dehydration with subsequent changes in the hydrogen-bonding
network. Changes in hydrogen-bonding patterns usually have an impact on the
global function of a biological system
- …
