We present a measurement of the mechanical properties of single polystyrene molecules of size R approximate to 50 nm. Using a micromechanical technique, we show directly that the polymer chain... Show moreWe present a measurement of the mechanical properties of single polystyrene molecules of size R approximate to 50 nm. Using a micromechanical technique, we show directly that the polymer chain behaves like a spring, and we measure the spring constant, We examine both cross-linked and non-cross-linked polystyrene. The former forms a stiffer spring, and the value of the spring constant is in agreement with theory, while in the latter case the measured value is larger than expected. Show less
The structural details of the metal site in the [His121]azurin mutant from Alcaligenes denitrificans where the axial methionine has been replaced by a histidine have been studied after substitution... Show moreThe structural details of the metal site in the [His121]azurin mutant from Alcaligenes denitrificans where the axial methionine has been replaced by a histidine have been studied after substitution with the divalent cadmium ion and the monovalent silver ion. The studies have been carried out in solution using the technique of perturbed angular correlations of gamma-rays (PAC) of the two isotopes, Ag-111 and Cd-111m.In the pH range 6-9, the PAC spectra for cadmium-substituted [His121]azurin reveals a pH-independent equilibrium between two different metal-coordination geometries. Interpretation of the PAC data shows agreement between the dominating coordination geometry and that derived from X-ray diffraction on the Cu(II)[His121] azurin at high pH (Messerschmidt, A., unpublished results). Thus: it appears likely that cadmium for this geometry is four coordinated to the ligands His46, His117: Cys112, and His121. The other geometry is best interpreted as a substitution of His121 by a solvent water ligand. These interpretations stem from predictions of the experimentally determined nuclear quadrupole interactions (NQI) via the simple angular overlap model (AOM). At low pH (3.8), the concentration of the former species is reduced to 50% of its high pH value suggesting a pK of about 4 for His121. Two different coordination geometries have also been observed for the Cu(II) protein and assigned a type 1.5 and a type 1 copper site [Kroes, S. J., Hoitink, C. W. G., Andrew, C. R., Ai, J., Sanders-Loehr, J., Messerschmidt, A., Hagen, W. R. & Canters, G. W. (1996a) fur: J. Biochem. 240, 342-351].For silver-substituted [His121]azurin, several notable changes occur relative to the cadmium-substituted protein. At least four different metal-coordination geometries exist for silver[His121]azurin in the pH range 4-8. Changes in the population of these coordination sites occurs between pK 4 and pH 5, and pK 5 and pH 6. Furthermore, in contrast to the cadmium-substituted protein, only a single coordination geometry is present above pH 6. The change in population occurring between pH 5 and pH 6 suggests ionization of a non-coordinating histidine, here proposed as His121. The change in population at low pH could then be due to protonation of an additional coordinating histidine such as His46 or His117. The single coordination geometry existing at pK values above 6 for the silver protein cannot within our model calculations be described with His121 coordinated. However, it can be described with a coordinated water molecule but in a different angular position than for His121 in the copper protein (Messerschmidt, A., unpublished results). The reduced tendency for silver to coordinate His121 is in agreement with the general trend of lower pK values for ligands coordinating to monovalent ions relative to divalent ions.In conclusion, this work demonstrates that mutation of Met121 to other amino acid residues opens the possibility of other coordination geometries than the rigid three-coordinated structure observed for wild-type azurin, especially the possibility of increasing the coordination number by either solvent water ligands or the substituting amino acid. Furthermore, it opens up the possibility for different coordination geometries for monovalent and divalent metal ions as observed here and previously for the [Leu121]azurin mutant [Bauer, R., Danielsen, E., Kemmingsen, L., Bjerrum, M. J., Hansson, ij. & Singh, K. (1997) J. Am. Chem. Sec. 119, 157-163]. Show less
Femtosecond transient absorption spectroscopy in the range of 500-1040 nm was used to study electron transfer at 5 K in reaction centers of Rhodobacter sphaeroides R26 in which the... Show moreFemtosecond transient absorption spectroscopy in the range of 500-1040 nm was used to study electron transfer at 5 K in reaction centers of Rhodobacter sphaeroides R26 in which the bacteriopheophytins (BPhe) were replaced by plant pheophytin a (Phe). Primary charge separation took place with a time constant of 1.6 ps, similar to that found in native RCs. Spectral changes around 1020 nm indicated the formation of reduced bacteriochlorophyll (BChl) with the same time constant, and its subsequent decay in 620 ps. This observation identifies the accessory BChl as the primary electron acceptor. No evidence was found for electron transfer to Phe, indicating that electron transfer from B-A(-) occurs directly to the quinone (Q(A)) through superexchange. The results are explained by a model in which the free energy level of P(+)Phe(-) lies above that of P+BA-, which itself is below P*. Assuming that the pigment exchange does not affect the energy levels of P* and P+BA-, our results strongly support a two-step model for primary electron transfer in the native bacterial RC, with no, or very little, admixture of superexchange. Show less
Bloemberg, G.V.; O'Toole, G.A.; Lugtenberg, E.J.J.; Kolter, R. 1997
The 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A.... Show moreThe 5' untranslated region (UTR) of the RNA of several tymoviruses contains conserved hairpins with protonatable internal loops, consisting of C-C and C-A mismatches (K. Hellendoorn, P. J. A. Michiels, R. Buitenhuis, and C. W. A. Pleij, Nucleic Acids Res. 24, 4910-4917, 1996). Here, we present a functional analysis of the 5' UTR of turnip yellow mosaic virus (TYMV) RNA, which contains two protonatable hairpins with nearly identical internal loops, Mutations were introduced in an infectious cDNA clone, and T7 RNA transcripts were used to infect Chinese cabbage plants, Different symptoms were observed for the various mutants, pointing to a functional role of the C-C and C-A mismatches in the hairpins of the 5' UTR. The replication of the virus is influenced by the mutations made, while in vitro translation studies showed that the expression of the two overlapping reading frames of TYMV is not influenced by the secondary structure of the leader. Various mutants were propagated for up to five serial passages of infection, and the sequence of the 5' UTR was determined. This resulted in virus RNA with new non-wild-type sequences that produced the wild-type phenotype in infected plants, Remarkably, in all cases C-C or C-A mismatches were introduced. The internal loop of the 5'-proximal hairpin seems to be more important for the viral life cycle than that of the second hairpin, A deletion of 75% of the leader, including the two hairpins, resulted in a virus that was deficient in viral spread. Since the ratio between filled and empty capsids was changed drastically by this mutation, a role of the 5' UTR in viral packaging is proposed. Show less
Rossum, B.J. van; Wachtveitl, J.; Raap, J.; Hoef, K. van der; Gast, P.; Lugtenburg, J.; ... ; Groot, H.J.M. de 1997
CP/MAS NMR data collected from L162YL mutant [4'-C-13]Tyr-enriched Rhodobacter sphaeroides RCs reveal that Tyr L162 is in a slightly heterogeneous and probably rigid section of the protein complex.... Show moreCP/MAS NMR data collected from L162YL mutant [4'-C-13]Tyr-enriched Rhodobacter sphaeroides RCs reveal that Tyr L162 is in a slightly heterogeneous and probably rigid section of the protein complex. The differences in chemical shifts of the individual components relative to those of the [4'-C-13]Tyr Rhodobacter sphaeroides R26 response are 0.2 ppm or less. This is small compared to the total dispersion of [4'-C-13] isotropic shifts, similar to 5 ppm, which measures the shift range due to variations in the microscopic environment between the various tyrosines in the protein complex. The structural changes in the mutant with respect to Rhodobacter sphaeroides R26, as probed by the labels, are thus minimal on the scale of the NMR. This suggests that the dramatic decrease of re-reduction rate of the oxidized primary donor P upon mutation (Farchaus et al., Biochemistry 32 (1993) 10885-10893) cannot be attributed to significant structural changes in the protein. Hence the NMR is in line with the current view that the decrease of the re-reduction rate in the mutant originates from slow reorientation of the docked cytochrome. (C) 1997 Elsevier Science B.V. Show less
By selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in alpha-crustacyanin, the major blue astaxanthin... Show moreBy selective isotope enrichment of astaxanthin, MAS NMR and semi-empirical modelling, ligand-protein interactions associated with the red shift in alpha-crustacyanin, the major blue astaxanthin binding carotenoprotein complex from the carapace of the lobster Homarus gammarus, have been analysed. C-13 Magic Angle Spinning (MAS) NMR spectra were obtained after reconstitution with astaxanthins labelled in the centre of the molecule or at the two keto groups. The MAS data reveal electrostatic polarizations of the conjugated chain. In addition, solid state NMR results for pure unlabelled astaxanthin can be compared with natural abundance C-13 MAS data for canthaxanthin and beta-carotene, to address the effect of the ring functionalities on the electronic properties of the polyene chain. Quantum chemical calculations were performed to reconcile the MAS data with one of several simple and straightforward mechanisms for the colour shift. The results point towards a colour shift mechanism in which the astaxanthin may be doubly charged, possibly by a double protonation of the two ring keto groups. Show less
The tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro.... Show moreThe tRNA-like structure at the 3' end of turnip yellow mosaic virus (TYMV) RNA was studied in order to determine the role of this structure in the initiation of minus-strand synthesis in vitro. Deletions in the 5'-to 3' direction up to the pseudoknot structure did not result in a decrease of transcription efficiency. However, transcription efficiency was reduced twofold when a fragment of 21 nucleotides, comprising the 3'-terminal hairpin, was used as a template, tRNA(Phe) from yeast, Escherichia coli 5S rRNA, and the 3'-terminal 208 nucleotides of alfalfa mosaic virus RNA 3 could not be transcribed by the RNA-dependent RNA polymerase (RdRp) of TYMV. Various mutations in the sequences of loop regions L1 and L2 or of stem region S1 of the pseudoknot were tested to further investigate the importance of the pseudoknot structure. The results were compared with those obtained in an earlier study on aminoacylation with the same mutants tR, M. W. Mans, M. H. van Steeg, P. W. G. Verlaan, C. W. A. Pleij, and L. Bosch. J. Mol. Biol, 223:221-232: 1992). Mutants which still harbor a stable pseudoknot, as proven by probing its structure, have a transcription efficiency very close to that of the wild-type virus. Disruption of the pseudoknot structure, however, gives rise to a drop in transcription efficiency to about 50%, No indications of base-specific interactions between L1, L2, or S1 of the pseudoknot and the RdRp were found. Show less
The quantum yield of the formation of the charge-separated state P(+)Q(A)(-) in reaction centers (RCs) of Rhodobacter sphaeroides R-26, in which the bacteriopheophytins in both the active (A) and... Show moreThe quantum yield of the formation of the charge-separated state P(+)Q(A)(-) in reaction centers (RCs) of Rhodobacter sphaeroides R-26, in which the bacteriopheophytins in both the active (A) and the inactive (B) branch are replaced by pheophytin (Pheo) a (Phi(A,B)-exchanged RCs), shows a positive temperature dependence: it is 38 +/- 5% between 10 and 60 K, increases with temperature to 72 +/- 5% at 200 K and shows a minor additional increase above this temperature. The temperature dependence of the quantum yield of P(+)Q(A)(-) formation in Phi(A,B)-exchanged RCs is modelled in the framework of a reaction scheme with the energy level of P(+)Pheo(A)(-) placed above P+BA- (Shkuropatov, A. Ya. and Shuvalov, V.A. (1993) FEES Lett. 322, 168-172), by the introduction of direct electron transfer from B-A(-) to Q(A), assisted by a superexchange-mechanism via P(+)Pheo(A)(-). The observed triples formation Phi(A,B)-exchanged RCs with pre-reduced Q(A) at cryogenic temperatures (quantum yield less than or equal to 12%) is attributed to a residual fraction of RCs in which only Phi(B) was exchanged for Pheo a. The lack of triplet formation in pre-reduced Phi(A,B)-exchanged RCs is consistent with our kinetic model, since this predicts that at low temperatures the state P(+)Pheo(A)(-) is not populated. (C) 1997 Elsevier Science B.V. Show less
In the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and. Met64 into Glu, The effect of this dipole on the electron-transfer... Show moreIn the hydrophobic patch of azurin from Pseudomonas aeruginosa, an electric dipole was created by changing Met44 into Lys and. Met64 into Glu, The effect of this dipole on the electron-transfer properties of azurin was investigated. From a spectroscopic characterization (NMR, EPR and ultraviolet-visible) it was found that both the copper site and the overall structure of the [Lys44, Glu64]azurin were not disturbed by the two mutations. A small perturbation of the active site at high pH, similar re, that observed for [Lys44]azurin. occurs in the double mutant. At neutral pH the electron-self-exchange rate constant of the double mutant shows a decrease of three orders of magnitude compared with the wild-type value. The possible reasons for this decrease are discussed. Electron transfer with the proposed physiological redox partners cytochrome c(551) and nitrite reductase have been investigated and the data analyzed in the Marcus framework. From this analysis it is confirmed that the hydrophobic patch of azurin is the interaction site with both partners, and that cytochrome c(551) uses its hydrophobic patch and nitrite reductase a negatively charged surface area for the electron transfer. Show less
![Coordination geometries for monovalent and divalent metal ions in [His121] azurin](/sites/all/modules/custom/islandora_solr_search/images/defaultimg.png?solr_nav%5Bid%5D=cfd98f5449ed0d094881&solr_nav%5Bpage%5D=0&solr_nav%5Boffset%5D=2)
![Electron-transfer properties of pseudomonas aeruginosa [Lys44, Glu64]azurin](/sites/all/modules/custom/islandora_solr_search/images/defaultimg.png?solr_nav%5Bid%5D=cfd98f5449ed0d094881&solr_nav%5Bpage%5D=0&solr_nav%5Boffset%5D=18)
