Biomedical subjects

H J de Groot

Publications and source records attributed to H J de Groot.

30 records · Page 2Linked to original sources

PubMed1997

13C Magic angle spinning NMR analysis and quantum chemical modeling of the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex in the carapace of the lobster Homarus gammarus.

Selective isotope enrichment, 13C magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze ligand-protein interactions associated with the bathochromic shift of astaxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crustacyanin were obtained after reconstitution with astaxanthins labeled with 13C at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12' in the complex, respectively. In contrast, at the 13 and 13' positions, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for positions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfield) and confirm the unequal perturbation of both halves after binding of the chromophore. However, these results also show that the main perturbation is of symmetrical origin, since the chemical shift differences exhibit a similar pattern in both halves of the astaxanthin molecule. A small downfield shift of 2.4 ppm was detected for the 4 and 4' positions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that charge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional groups, as models of ligand-protein interactions. Taking into account the color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The results point toward a mechanism in which the astaxanthin is charged and subject to strong electrostatic polarizations originating from both keto groups, most likely a double protonation.

Animals↗

PubMed1997

Are recommendations from carcinoma of the cervix Patterns of Care Studies (PCS) in the United States of America (USA) applicable to centers in developing countries?

PURPOSE: To compare patient demographics, treatment resources, practice patterns, and outcome results for squamous cell carcinoma of the uterine cervix (SCC) between the 1978 and 1983 Patterns of Care studies (PCS) in the United States of America (USA) and a nonacademic center within a developing country. METHOD AND MATERIALS: Patient details (race, age, stage, and number per year), treatment used, and treatment outcome were retrieved from the charts of the 1160 cases registered at this center with SCC of the cervix between 1976 and 1985. Demographic variables and Kaplan-Meier survival estimates were calculated and compared with results from published PCS reviews. RESULTS: There is a significant difference in the racial group presentation of cervix cancer at this center compared with the PCS reviews (p < 0.005), and median ages are significantly lower at this center (t = p < 0.001). The proportion of patients with Stage III or more was significantly higher at this center than the PCS centers (24 vs. 47%, p < 0.001). There were also vast differences in facility resources. Fewer cases at this center underwent intracavitary insertions than at PCS centers. Mean Point A doses were significantly reduced for this center compared with the PCS reviews. Kaplan-Meier estimates were similar for Stage I and II in PCS centers and this center, but were inferior for this center in Stage III patients (p < 0.05 for OS and p < 0.01 for LC). Late morbidity rates were similar for both PCS centers and this center. CONCLUSION: PCS recommendations may be applicable to nonacademic centers within developing countries, if the latter use staging techniques that are consistent with the PCS staging guidelines.

Brachytherapy↗

PubMed1995

CP-MAS 13C-NMR dipolar correlation spectroscopy of 13C-enriched chlorosomes and isolated bacteriochlorophyll c aggregates of Chlorobium tepidum: the self-organization of pigments is the main structural feature of chlorosomes.

Magic angle spinning (MAS) NMR dipolar correlation spectroscopy was applied for the first time to a biologically intact system, the light-harvesting chlorosomes of the green photosynthetic bacterium Chlorobium tepidum. The MAS spectra provide evidence that the self-organization of many thousands of bacteriochlorophyll c (BChl c) molecules is the predominant structural feature of the chlorosome. 13C-Enriched chlorosomes were prepared from nonuniformly labeled cultures grown with NaH13CO3 as the main carbon source and from a uniformly 13C-labeled culture grown with NaH13CO3 as the sole carbon source. For the nonuniformly labeled samples, the positions of the chlorin macrocycle originating from C-4 and C-5 of 5-aminolevulinic acid contained > 95% 13C while the remaining positions, which could have originated also from unlabeled acetate, were labeled to approximately 60% with 13C. The 1-D and 2-D MAS data of the labeled chlorosomes, when compared with data on the isolated labeled BChl c aggregated in n-hexane, show that the major component of the MAS signals in the chlorosomes is from BChl c, and only minor signal contributions arise from lipids and proteins. The 13C MAS signals of the BChl c aggregates were fully assigned by MAS 2-D dipolar correlation spectroscopy, using data on monomeric BChl c in CDCl3/CD3OD as reference. The 2(1)-, 3-, 3(2-), 5-, 12(1)-, 13-, and 13(1)-carbons are shifted by 2.5 ppm or more upfield with respect to the solution data.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminolevulinic Acid↗

PubMed1995

13C magic angle spinning NMR characterization of the functionally asymmetric QA binding in Rhodobacter sphaeroides R26 photosynthetic reaction centers using site-specific 13C-labeled ubiquinone-10.

Photosynthetic reaction centers (RCs) of Rhodobacter sphaeroides R26 were reconstituted at the QA site with ubiquinone-10, selectively 13C-enriched on positions 1, 2, 3, 4, and 3-Me (IUPAC numbering). RCs dispersed in LDAO detergent were studied with 13C CP/MAS NMR spectroscopy at temperatures between 180 and 240 K, while RCs precipitated by removal of the detergent were investigated at ambient temperature and at temperatures down to 180 K. Electrostatic charge differences in QA induced by polarization from the protein are less than 0.02 electronic equivalent for any of the labeled positions. This includes the 4-carbonyl, which is therefore not significantly polarized by an electrostatic binding interaction with the protein. The QA site is slightly heterogeneous on the scale of the NMR as the observed line widths of the labels are between 150 and 300 Hz and inhomogeneous broadening is observed for the signals of positions 1, 2, and 3 upon cooling. This contrasts with earlier MAS observations for labels in the vicinity of the special pair. The chemical shifts are 184, 144, and 137 ppm for the labels at positions 1, 2, 3, and 12 ppm for the 3-methyl 13C. For the 4-carbonyl only at sample temperatures below approximately 255 K a CP/MAS response can be observed at 183 ppm. The principal components of the chemical shift tensors for the ring labels in QA were estimated using difference spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗

PubMed1995

FTIR spectroscopy shows weak symmetric hydrogen bonding of the QB carbonyl groups in Rhodobacter sphaeroides R26 reaction centres.

The absorption frequencies of the C = O and C = C (neutral state) and of the C...O (semiquinone state) stretching vibrations of QB have been assigned by FTIR spectroscopy, using native and site-specifically 1-, 2-, 3- and 4-13C-labelled ubiquinone-10 (UQ10) reconstituted at the QB binding site of Rhodobacter sphaeroides R26 reaction centres. Besides the main C = O band at 1641 cm-1, two smaller bands are observed at 1664 and 1651 cm-1. The smaller bands at 1664 and 1651 cm-1 agree in frequencies with the 1- and 4-C = O vibrations of unbound UQ10, showing that a minor fraction is loosely and symmetrically bound to the protein. The larger band at 1641 cm-1 indicates symmetric H-bonding of the 1- and 4-C = O groups for the larger fraction of UQ10 but much weaker interaction as for the 4-C = O group of QA. The FTIR experiments show that different C = O protein interactions contribute to the factors determining the different functions of UQ10 at the QA and the QB binding sites.

Binding Sites↗

PubMed1995

Protein-chromophore interactions in alpha-crustacyanin, the major blue carotenoprotein from the carapace of the lobster, Homarus gammarus. A study by 13C magic angle spinning NMR.

MAS (magic angle spinning) 13C NMR has been used to study protein-chromophore interactions in alpha-crustacyanin, the blue astaxanthin-binding carotenoprotein of the lobster, Homarus gammarus, reconstituted with astaxanthins labelled with 13C at the 14,14' or 15,15' positions. Two signals are seen for alpha-crustacyanin containing [14,14'-13C2]astaxanthin, shifted 6.9 and 4.0 ppm downfield from the 134.1 ppm signal of uncomplexed astaxanthin in the solid state. With alpha-crustacyanin containing [15,15'-13C2]astaxanthin, one essentially unshifted broad signal is seen. Hence binding to the protein causes a decrease in electronic charge density, providing the first experimental evidence that a charge redistribution mechanism contributes to the bathochromic shift of the astaxanthin in alpha-crustacyanin, in agreement with inferences based on resonance Raman data [Salares, et al. (1979) Biochim. Biophys. Acta 576, 176-191]. The splitting of the 14 and 14' signals provides evidence for asymmetric binding of each astaxanthin molecule by the protein.

Animals↗

PubMed1994

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1-, 2-, 3- and 4-13C site-specifically labelled ubiquinone-10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra-red bands dominated by the 1- and 4-C = O vibration of QA are assigned in the QA(-)-QA difference spectra. The mode dominated by the 4-C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone-10. In contrast, the mode dominated by the 1-C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4-C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge-separated state the mode dominated by the semiquinone 4-C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1-C = O vibration indicates a weaker interaction. The asymmetric binding of the 1- and 4-C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone-10 at the QA site as compared with its reactions at the QB site.

Binding Sites↗

PubMed1992

13C magic angle spinning NMR evidence for a 15,15'-cis configuration of the spheroidene in the Rhodobacter sphaeroides photosynthetic reaction center.

The photosynthetic reaction center of Rhodobacter sphaeroides 2.4.1 contains one carotenoid that protects the protein complex against photodestruction. The structure around the central (15,15') double bond of the bound spheroidene carotenoid was investigated with low-temperature magic angle spinning 13C NMR, which allows an in situ characterization of the configuration of the central double bond in the carotenoid. Carotenoidless reaction centers of R. sphaeroides R26 were reconstituted with spheroidene specifically labeled at the C-14' or C-15' position, and the signals from the labels were separated from the natural abundance background using 13C MAS NMR difference spectroscopy. The resonances shift 5.2 and 3.8 ppm upfield upon incorporation in the protein complex, similar to the 5.6 and 4.4 ppm upfield shift occurring in the model compound beta-carotene upon trans to 15,15'-cis isomerization. Hence the MAS NMR favors a cis configuration, as opposed to the trans configuration deduced from X-ray data.

Carbon Isotopes↗

PubMed1992

13C magic angle spinning NMR study of the light-induced and temperature-dependent changes in Rhodobacter sphaeroides R26 reaction centers enriched in [4'-13C]tyrosine.

Solid-state 13C magic angle spinning (MAS) NMR has been used to investigate detergent-solubilized photosynthetic reaction centers of Rhodobacter sphaeroides R26, selectively enriched in [4-13C]-tyrosine. The reaction centers were frozen, in the dark and while subject to intense illumination, and studied at temperatures between approximately 215 and approximately 260 K. The signal consists of at least seven narrow lines superimposed on a broad doublet. The chemical shift anisotropy is similar to that for crystalline tyrosine. The two narrowest resonances, corresponding to signals from individual tyrosines, are 28 +/- 5 Hz wide, comparable to what is observed for quaternary carbons in linearly elastic organic solids. The line width as well as the chemical shift of these signals is essentially independent of temperature. This provides strong evidence for an unusually ordered, well-shielded, and structurally, electrostatically, and thermodynamically stable interior of the protein complex without structural heterogeneities. As the temperature is lowered, additional signal from the labels develops and the natural abundance resonances from the detergent broaden, providing evidence for considerable flexibility at the exterior of the protein complex and in the detergent belt at the higher temperatures. In addition, the NMR provides evidence for an electrostatically uniform and neutral complex, since the total dispersion in isotropic shifts for the labels is < 5 ppm and corresponds to electron density variations of less than 0.03 electronic equivalents with respect to tyrosine in the solid state or in solution. When the sample is frozen while subject to intense illumination, a substantial part of the protein is brought into the charge-separated state P.+QA.-. At least three sharp resonances, including the narrowest lines, are substantially reduced in intensity. It is argued that this effect is caused by the electronic spin density associated with the oxidized primary donor P.+. These results strongly suggest that the environment of the special pair is extremely rigid and question the role of protein conformational distortions during the primary photoprocess.

Carbon Isotopes↗

PubMed1990

Solid-state 13C and 15N NMR study of the low pH forms of bacteriorhodopsin.

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.

Bacteriorhodopsins↗

PubMed1989

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR.

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Adaptation, Ocular↗

PubMed1989

Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: counterion effects on the 15N shift anisotropy.

High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shift anisotropy for the Schiff base nitrogen in bR548 and bR568. Comparisons are made between all-trans-bR568 and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in 15N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR568 follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.

Bacteriorhodopsins↗