Biomedical subjects
Publications and source records attributed to M E Peek.
Here we have stressed important differences between protein and DNA crystallography. Crystal growth and data collection methodologies are not directly transferable between the two subfields. In addition, we note that analysis of symmetry and packing of DNA crystals can be useful and a uniquely aesthetic exercise.
We characterize intercalative complexes as either "high charge" and "low charge". In low charge complexes, stacking interactions appear to dominate stability and structure. The dominance of stacking is evident in structures of daunomycin, nogalamycin, ethidium, and triostin A/echinomycin. By contrast in a DNA complex with the tetracationic metalloporphyrin CuTMPyP4 [copper (II) meso-tetra(N-methyl-4-pyridyl)porphyrin], electrostatic interactions appear to draw the porphyrin into the duplex interior, extending the DNA along its axis, and unstacking the DNA. Similarly, DNA complexes of tetracationic ditercalinium and tetracationic flexi-di show significant unstacking. Here we report x-ray structures of complexes of the tetracationic bis-intercalator D232 bound to DNA fragments d(CGTACG) and d(BrCGTABrCG). D232 is analogous to ditercalinium but with three methylene groups inserted between the piperidinium groups. The extension of the D232 linker allows it to sandwich four base pairs rather than two. In comparison to CuTMPyP4, flexi-di and ditercalinium, stacking interactions of D232 are significantly improved. We conclude that it is not sufficient to characterize intercalators simply by net charge. One anticipates strong electrostatic forces when cationic charge is focused to a small volume or region near DNA and so must consider the extent to which cationic charge is focused or distributed. In sum, ditercalinium, with a relatively short linker, focuses cationic charge more narrowly than does D232. So even though the net charges are equivalent, electrostatic charges are expected to be of greater structural significance in the ditercalinium complex than in the D232 complex.
We report the 2.4 A resolution X-ray structure of a complex in which a small molecule flips a base out of a DNA helical stack. The small molecule is a metalloporphyrin, CuTMPyP4 [copper(II) meso-tetra(N-methyl-4-pyridyl)porphyrin], and the DNA is a hexamer duplex, [d(CGATCG)]2. The porphyrin system, with the copper atom near the helical axis, is located within the helical stack. The porphyrin binds by normal intercalation between the C and G of 5' TCG 3' and by extruding the C of 5' CGA 3'. The DNA forms a distorted right-handed helix with only four normal cross-strand Watson-Crick base pairs. Two pyridyl rings are located in each groove of the DNA. The complex appears to be extensively stabilized by electrostatic interactions between positively charged nitrogen atoms of the pyridyl rings and negatively charged phosphate oxygen atoms of the DNA. Favorable electrostatic interactions appear to draw the porphyrin into the duplex interior, offsetting unfavorable steric clashes between the pyridyl rings and the DNA backbone. These pyridyl-backbone clashes extend the DNA along its axis and preclude formation of van der Waals stacking contacts in the interior of the complex. Stacking contacts are the primary contributor to stability of DNA. The unusual lack of van der Waals stacking contacts in the porphyrin complex destabilizes the DNA duplex and decreases the energetic cost of local melting. Thus extrusion of a base appears to be facilitated by pyridyl-DNA steric clashes.
We have determined the x-ray structure of a DNA fragment containing 7,8-dihydro-8-oxoguanine (G(O)). The structure of the duplex form of d(CCAGOCGCTGG) has been determined to 1.6-A resolution. The results demonstrate that GO forms Watson-Crick base pairs with the opposite C and that G(O) is in the anti conformation. Structural perturbations induced by C.G(O)anti base pairs are subtle. The structure allows us to identify probable elements by which the DNA repair protein MutM recognizes its substrates. Hydrogen bond donors/acceptors within the major groove are the most likely element. In that groove, the pattern of hydrogen-bond donors/acceptors of C.G(O)anti is unique. Additional structural analysis indicates that conversion of G to G(O) would not significantly influence the glycosidic torsion preference of the nucleoside. There is no steric interaction of the 8-oxygen of G(O) with the phospho-deoxyribose backbone.
The bis-intercalator ditercalinium (NSC 366241), composed of two 7 H-pyridocarbazoles linked by a bis(ethylpiperidinium), binds to DNA with a binding constant greater than 10(7) M-1. One distinctive aspect of the 3-D X-ray structure of a DNA-ditercalinium complex is its asymmetry. We propose here that the activity of ditercalinium may be related to structural polymorphism and dynamic conversion between conformers. It was previously reported that activity is closely related to linker composition. Activity increases with increasing conformational restraints of the linker. We suggest these conformational restraints can lead to asymmetry in DNA complexes and that this asymmetry results directly in structural polymorphism. Using the Cambridge Structural Database (CSD) as a source of information about chemical fragments that are analogous to the linker of ditercalinium, we have explored the conformational space available to ditercalinium. The results indicate that the linker is highly constrained and that the DNA complex is intrinsically asymmetric. We propose a reasonable mechanism of ring reversal that is consistent with the conformations of analogous fragments within the CSD.
The bis-intercalators Flexi-Di and ditercalinium are synthetic dimers that bis-intercalate into DNA and cause cell death in prokaryotes from futile and abortive repair of DNA. Each is composed of two 7H-pyridocarbazole units and a linker. Flexi-Di has a flexible spermine-like linker while ditercalinium has a rigid bis(ethylpiperidinium) linker. This report, describing the 2.5-A X-ray structure of Flexi-Di complexed with [d(BrCGCG)]2, appears to be the first report of a three-dimensional structure of a DNA complex with a bis-intercalator with a flexible linker. DNA complex formation with a ditercalinium analog having a flexible linker was not anticipated to yield unstacked and bent DNA as was observed in the previously reported ditercalinium.[d(CGCG)]2 complex. Surprisingly, the DNA in the Flexi-Di complex is bent to a degree exceeding that of the ditercalinium complex. A comparison of the DNA complexes of Flexi-Di and ditercalinium has allowed us to propose a mechanism by which these bis-intercalators distort DNA. We propose that this class of bis-intercalators pulls the internal base pairs into the major groove and pushes the external base pairs into the minor groove. The result is a bend toward the minor groove. It appears that hydrogen bonds between the linker and the internal guanines effectively pull the central base pairs of the complex out into the major groove. At the external regions of the complex, stacking interactions between the chromophores and terminal base pairs effectively push the terminal base pairs into the minor groove. The result of this push/pull combination is to bend the DNA.
In crystallographic structures of biological macromolecules, one can observe many hydration rings that originate at one water molecule, pass via hydrogen bonds through several others, and return to the original water molecule. Five-membered water rings have been thought to occur with greater frequency than other ring sizes. We describe a quantitative assessment of relationships between water ring size and frequency of occurrence in the vicinity of nucleic acid interfaces. This report focuses on low-temperature X-ray crystallographic structures of two anthracyclines, adriamycin (ADRI) and daunomycin (DAUN), bound to d(CGATCG) and on several DNA structures published previously by others. We have obtained excellent low-temperature (-160 degrees C, LT) X-ray intensity data for d(CGATCG)-adriamycin and d(CGATCG)-daunomycin with a multiwire area detector. The LTX-ray data sets contain 20% (daunomycin, LT-DAUN) and 35% (adriamycin, LT-ADRI) more reflections than were used to derive the original room-temperature (15 degrees C) structures [Frederick, C.A., Williams, L.D., Ughetto, G., van der Marel, G. A., van Boom, J.H., Rich, A., & Wang, A.H.-J. (1990) Biochemistry 29, 2538-2549]. The results show that five-membered water rings are not preferred over other ring sizes. This assessment is consistent with our observation of broad dispersion W-W-W angles (sigma = 20 degrees). In addition, we report that the thermal mobility, distinct from the static disorder, of the amino sugar of daunomycin and adriamycin is significantly greater than that of the rest of the complex. This mobility implies that if the central AT base pair is switched to a CG base pair, there should be a low energy cost in avoiding the guanine amino group. The energy difference (for the sugar-binding preference) between d(CGTACG) and d(CGCGCG) could be considerably less than 20 kcal/mol, a value proposed previously from computation.
At 9 wk postpartum the difference in energy intake of 40 lactating (2440 +/- 430 kcal/d) and 16 nonlactating women (1680 +/- 400 kcal/d) was 760 kcal/d but decreased to 550 kcal/d when adjusted for habitual intakes and body weight. Energy cost of lactation amounted to 650 kcal/d (breast-milk production, 745 +/- 130 g/d). When compared with nonlactating counterparts, the lactating women mainly achieved energy balance by eating more. Sixteen of the 40 lactating women were also studied at 56 wk. Their cost of lactation at 5-13 wk was 630 kcal/d (breast-milk production, 720 +/- 124 g/d); these women met their energy cost of lactation by eating more (415 kcal/d); by tissue mobilization (35 kcal/d), and by reducing energy expenditure (180 kcal/d). The present study helps in the understanding of how well-nourished women with an adequate lactational performance may cope in everyday life with the energy stress of lactation, and suggests that current recommendations of energy needs during lactation are too high.
Body weight, basal metabolic rate (BMR), and treadmill metabolic rate (TMR) (3.9 km/h, no elevation) were measured in 39 women at 12, 24, and 36 wk gestation and at 9 wk postpartum. Prepregnancy measurements were also made on 15 of the women. TMR at 36 wk (3.65 +/- 0.50 kcal/min) was significantly higher than at 24 wk (3.38 +/- 0.43 kcal/min) or at 9 wk postpartum (3.38 +/- 0.43 kcal/min). Net energy cost (TMR minus BMR) at 36 wk gestation (2.42 +/- 0.40 kcal/min) was not different from prepregnancy or postpartum values but was significantly higher than at 12 wk (2.28 +/- 0.39 kcal/min) and 24 wk (2.28 +/- 0.37 kcal/min) gestation. In eight women the energy cost of self-paced walking on a treadmill was measured. The absolute and net energy cost decreased sharply from 6 to 12 wk gestation (by 8% and 11%, respectively) but remained unchanged afterwards. The data suggest that in the energy requirements for pregnant women no additional allowance need be made for physical activity, even if a woman's activity pattern includes a substantial amount of externally paced work.
Basal metabolic rate (BMR), activity pattern, and energy costs of some daily activities were measured in 25 Dutch women throughout pregnancy and the first year postpartum. Physical activity index (PAI), which refers to daily energy expenditure expressed as a multiple of BMR, was calculated from activity-pattern data and activity costs. Mean PAIs (+/- SD) throughout pregnancy, during the first 6 mo postpartum, and at 1 y postpartum were 1.48 +/- 0.08, 1.49 +/- 0.07, and 1.53 +/- 0.10 X BMR, respectively. Because measured BMR at 1 y postpartum was 1440 +/- 168 kcal/d, costs for physical activity in pregnancy and the first 6 mo postpartum were, respectively, approximately 70 and approximately 50 kcal/d lower than at 1 y postpartum. For women with sedentary lifestyles the energy saved during pregnancy and lactation because of decreased physical activity and decreased costs of activities will be limited.
Body weight, fat mass, and basal metabolic rate were measured longitudinally from early pregnancy until 2 mo postpartum in 57 healthy Dutch women; 23 of whom were also studied before pregnancy. Weight gain over pregnancy was 11.8 +/- 3.7 kg and weight gain from 12 wk gestation to delivery was 10.3 +/- 3.8 kg. Birth weights and placental weights were 3458 +/- 527 and 657 +/- 114 g, respectively. Gain in maternal fat stores over pregnancy was 2.0 +/- 2.6 kg and difference in fat mass from 12 wk gestation to 5 wk postpartum was 1.2 +/- 2.2 kg. The energy equivalent of the gain in fat stores, including costs of synthesizing, can be estimated as 22,000 kcal. The cumulative increment in basal metabolism over pregnancy was 34,350 +/- 30,000 kcal. When the energy equivalent of the gain in tissue other than fat stores is assumed to be approximately 11,750 kcal, total energy cost of pregnancy is at 68,100 +/- 38,560 kcal.
The equations for estimating fat mass from body density or total body water are not appropriate for application in pregnancy, because the underlying assumptions with respect to density and composition of fat-free mass do not hold for pregnancy. Representative values have been derived from literature data for density and water content of maternal fat-free mass throughout pregnancy. Using these values we developed a method that provides new equations for estimating fat mass from body density or total body water for any desired stage of pregnancy. The validity of the new equations based on body density is discussed using data on body weight and body density obtained from a longitudinal study on well-nourished Dutch pregnant women. Because the new equations result in more valid estimates of maternal body fat mass, we suggest that they be used in studies on energy balance in pregnancy.
57 healthy Dutch women were studied longitudinally from early pregnancy until 2 months post partum. Regular measurements were made of energy intake in food, basal metabolic rate, body weight and body fat mass, and levels of physical activity. Some data were obtained before conception in 23 women. The energy cost of pregnancy calculated as the energy deposited as new tissues plus the associated increase in basal metabolism amounted to 286 MJ (1020 kJ/day), which is only 11% lower than the theoretical estimate of requirements of 323 MJ (1 MJ = 239 kcal). Energy intake throughout the first 10 wk of pregnancy was identical to that before pregnancy. Energy intake was only 200 kJ/day higher in late than in early pregnancy (not significant), and the cumulative increase in energy intake over pregnancy was estimated as 22 MJ (about 80 kJ/day). There is, therefore, an energy gap in pregnancy of about 940 kJ/day. It is proposed that the main mechanisms by which the pregnant body is able to save energy and to bridge the energy gap are by adjustments to physical activity and an increase in work efficiency and an adaptation of the metabolic response to food. Savings on physical activity by behavioural adaptations will not exceed 355 kJ/day.