The binding of the primary quinone anion radical Q(A)(-.) in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides species was investigated with electron spin echo envelope modulation (ESEEM).... Show moreThe binding of the primary quinone anion radical Q(A)(-.) in reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides species was investigated with electron spin echo envelope modulation (ESEEM). The ESEEM spectra, at two microwave frequencies, of Zn-substituted RCs of Rb. sphaeroides R26 showed interactions of the unpaired electron of Q(A)(-.) with two nitrogen nuclei in the protein matrix. From an analysis of the experimental data the nitrogen nuclear quadrupole resonance parameters were determined: e(2)qQ/h = 1.52 MHz, eta = 0.82 and e(2)qQ/h = 3.04 MHz, eta = 0.66, which are assigned to the N-14(8(1))-H group of His M219 and the peptide N-14 of Ala M260, The ESEEM spectrum of Q(A)(-.) in reaction centers of the Rb, sphaeroides mutant W(M252)Y shows that the nitrogen of Trp M252 is not interacting with Q(A)(-.), that of the mutant H(M266)C shows that manipulating the Fe binding site strongly affects the Q(A)-binding site. Show less
The secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison, Despite large... Show moreThe secondary structures of the 5'-untranslated region (5'-UTR) of five different tymoviruses have been determined by structure probing, computer prediction and sequence comparison, Despite large sequence differences, there are remarkable similarities in the secondary structure, In all viruses two or four hairpins are found, most of which contain a symmetrical internal loop consisting of adjacent C-C or C-A mismatches, Since it is known that such mismatches can be protonated and protonated cytosines play an important role in RNA-protein interactions in tymoviral virions, the influence of pH on the conformation of the internal loop was studied, UV melting experiments and 1-dimensional proton NMR at varying pH values and salt concentrations confirm that the hairpins can be protonated under relatively mild conditions, The hairpin found in the 5'-UTR of erysimum latent virus, which has an asymmetrical internal loop consisting of cytosines and uridines, shows comparable behaviour, It is concluded that all tymoviral RNAs contain protonatable hairpins in the 5'-UTR, Binding experiments with empty viral capsids, however, do not yet establish a role in capsid protein binding. Show less
The interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different... Show moreThe interaction of water molecules with copper in wild-type azurin and different site-directed mutants of the coordinated residues is studied by nuclear magnetic relaxation dispersion. Different degrees of solvent accessibility are found. The low relaxivity of wild-type azurin agrees with a solvent-protected copper site in solution, the closest water being found at a distance of more than 5 Angstrom from the copper. This low relaxivity contrasts with the relatively large relaxivity of the His46Gly and His117Gly azurin mutants, which shows clear evidence of copper-coordinated water. The data on the latter mutants are best analyzed in terms of one and two water molecules coordinated to the copper in His46Gly and His117Gly, respectively. The Met121His azurin mutant shows an intermediate behavior. The data are analyzed in terms of an increased solvent accessibility with respect to the wild-type azurin, resulting in semi-coordination of water at low pH. These different modes of coordination lead to different geometries, ranging from the trigonal type 1 site of b wild-type azurin to the tetragonal type 2 copper sites of the His117Gly and His46Gly azurin mutants through a so-called type 1.5 site of the Met121His mutant. A correlation is found between the relaxation time (tau(s)) of the unpaired electron of copper(II) and the geometry of the metal site: as the tetragonal character decreases the relaxation becomes significantly faster. tau(s) values of less than or equal to 1 ns are found for the tetrahedrally distorted type 1 and type 1.5 sites and of 5-15 ns for the tetragonal type 2 sites. Show less
Radicals produced by illumination or ionizing radiation are important in life sciences and chemistry. Reducing the rate of radical decay by recombination can be vital for maximizing the yield of... Show moreRadicals produced by illumination or ionizing radiation are important in life sciences and chemistry. Reducing the rate of radical decay by recombination can be vital for maximizing the yield of radical pair products, or to study reaction pathways. We propose a new method to reduce the probability for singlet-born radical pair recombination, based on the population of energetically isolated radical pair spin levels, and present an application to photoinduced radical pairs in photosynthetic reaction centers, for which we have achieved a 40-fold increase in the low temperature (23 K) radical pair lifetime. Show less
The UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two... Show moreThe UV dynamic fluorescence and CD of several Pseudomonas aeruginosa azurins bearing single amino acid mutation have been studied. Two classes of mutants were examined. In the first class, two hydrophobic residues in the core of the protein, Ile 7 and Phe 110, nearest to the azurin single tryptophan Trp 48, were substituted by a serine (mutants I7S and F110S). In the second class, two residues in the outer sphere of the copper ligand field were changed, obtaining the following mutants: M44K, H35F; H35L, and H35Q, All these proteins showed two fluorescence lifetimes in the copper-containing form, but only one in the copper-free form. The lifetime of the latter derivatives was different from either those of the metal-bound samples, definitely ruling out the presence of apo-like species in the hole protein. Copper-Free I7S and F110S showed a more complex fluorescence decay profile requiring a distribution of lifetimes rather than a single lifetime. Holo F110S was also better fitted, in the limit of confidence, with two distributions rather than a pair of lifetimes. Time-resolved anisotropy of these two mutants as well as of wild-type (wt) protein showed two components (rotational times for wt less than or equal to 200 ps and 7 ns, respectively). These components were not affected significantly by copper removal in the case of wt protein. Instead, the short rotational component of the mutants dropped dramatically to values near zero, indicating a much greater mobility of the tryptophanyl residue in the mutant apo azurins, These data were supported by CD measurements showing a small effect of the copper presence in the region below 250 nm, i.e., in the secondary structure, but almost a collapse of the aromatic asymmetry at 270-295 nm related to a relaxation of the structural constraint around the tryptophan. Altogether these data show that copper does not play a structural role in wt azurin, whereas it is crucial in the stabilization of I7S and F110S mutants. Furthermore, although the metal site geometry is rigidly kept in wt apo-azurin, it regains the native form only in the presence of the metal in the ''core'' mutants. This finding is important for the theory of entatic states in metalloproteins Show less
We report Shpol'skii-type luminescence spectra of C-70 in n-pentane at 1.5 K with vibronic linewidths of 3 cm(-1). The vibronic structure of both fluorescence and phosphorescence has been... Show moreWe report Shpol'skii-type luminescence spectra of C-70 in n-pentane at 1.5 K with vibronic linewidths of 3 cm(-1). The vibronic structure of both fluorescence and phosphorescence has been unravelled. The fluorescence intensity largely shows up in vibronic bands corresponding to transitions to ground-state infrared-active vibrational modes and it is concluded that the first excited singlet state of C-70 in alkanes is of A'(2) character. Show less
Dennison, C.; Berg, A.; Vries, S. de; Canters, G.W. 1996
The dinuclear paramagnetic center of the soluble Cu-A domain of the cytochrome c oxidase from Bacillus subtilis has been studied using H-1 NMR. The spectrum possesses remarkably sharp shifted... Show moreThe dinuclear paramagnetic center of the soluble Cu-A domain of the cytochrome c oxidase from Bacillus subtilis has been studied using H-1 NMR. The spectrum possesses remarkably sharp shifted resonances, Comparison with the spectrum of the Cu-A amicyanin variant provides the spin density distribution in the Cu-A site of cytochrome c oxidase. This represents the first paramagnetic NMR study of the dinuclear Cu-A center from the soluble domain of subunit II of cytochrome c oxidase. Show less
Microperoxidases with increasing lengths of the peptide attached to the heme moiety have been isolated after proteolytic digestion of horse-heart cytochrome c (microperoxidases 6, 8, and 11) and of... Show moreMicroperoxidases with increasing lengths of the peptide attached to the heme moiety have been isolated after proteolytic digestion of horse-heart cytochrome c (microperoxidases 6, 8, and 11) and of cytochrome c(550) from Thiobacillus versutus (microperoxidase 17). The different microperoxidases catalyze the H2O2-dependent para-hydroxylation of aniline relatively efficiently but are rapidly inactivated under turnover conditions. The horse-heart cytochrome-c-derived microperoxidases have identical values for V-max but show a decrease of the K-m for aniline and a higher stability when the attached peptide is longer. The kinetic constants obtained for microperoxidase 17, differ markedly from the microperoxidases derived from horse-heart cytochrome c. Possible factors underlying the observed differences are discussed. Show less
To examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which... Show moreTo examine the function of the central pseudoknot in 16S rRNA, we have studied Escherichia coli 30S subunits with the A(18) mutation in this structure element, Previously, this mutation, which changes the central base pair of helix 2, C-18-G(917) to an A(18)xG(917) mismatch, was shown to inhibit translation in vivo and a defect in initiation was suggested, Here, we find that the mutant 30S particles are impaired in forming 70S tight couples and predominantly accumulate as free 30S subunits, Formation of a 30S initiation complex, as measured by toeprinting, was almost as efficient for mutant 30S subunits, derived from the tight couple fraction, as for the wild-type control, However, the A(18) mutation has a profound effect on the overall stability of the subunit, The mutant ribosomes were inactivated by affinity chromatography and high salt treatment, due to easy loss of ribosomal proteins, Accordingly, the particles could be reactivated by partial in vitro reconstitution with 30S ribosomal proteins, Mutant 30S subunits from the free subunit fraction were already inactive upon isolation, but could also be reactivated by reconstitution, Apparently, the inactivity in initiation of these mutant 30S subunits is, at least in part, also due to the lack of essential ribosomal proteins, We conclude that disruption of helix 2 of the central pseudoknot by itself does not affect the formation of a 30S initiation complex, We suggest that the in vivo translational defect of the mutant ribosomes is caused by their inability to form 70S initiation complexes. Show less
The RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of... Show moreThe RNA of all tymoviruses, a group of ssRNA plant viruses, has a base composition that is different from that of most other viruses. The excess of cytosines (35-42%) and the low number of guanosines (15-17%) must impel an RNA structure with a relatively low amount of base pairing and a high incidence of unpaired cytosines. These unpaired cytosines probably function in RNA-protein interactions. To gain insight into the way the RNA is positioned inside the virion, the secondary structure has been determined of a part of TYMV RNA, including the so-called tymobox, the coat protein gene, and the 3' untranslated region, by structure probing, sequence comparison, and computer predictions. Conservation of secondary structure elements in tymoviruses is not high and does not parallel the conservation of the primary structure. A combination of structure prediction and probing experiments, however, results in a model consisting of structured domains of 100-200 nucleotides interspersed by long unpaired cytosine-rich regions. The latter may interact with the coat protein inside the virion. The structure of some functionally interesting regions of the 3' part of TYMV RNA is also discussed. (C) 1996 Academic Press, Inc. Show less
Bush beloofde de wereld een nieuwe orde. Natuurlijk kwam hier niets van terecht. Uiteraard wordt er nog overal op de wereld gevochten, volgens sommigen meer dan voorheen. Maar dat ligt... Show moreBush beloofde de wereld een nieuwe orde. Natuurlijk kwam hier niets van terecht. Uiteraard wordt er nog overal op de wereld gevochten, volgens sommigen meer dan voorheen. Maar dat ligt waarschijnlijk aan het feit dat we nu veel beter op de hoogte zijn, dankzij de televisie, radio en krant. Show less
