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CO Rebinding to Protoheme: Investigations of the Proximal and Distal Contributions to the Geminate Rebinding Barrier
Ye, Xiong
Yu, Anchi
Georgiev, Georgi Y.
Gruia, Florin
Ionascu, Dan
Cao, Wenxiang
Sage, J. Timothy
Champion, Paul M.
Journal of the American Chemical Society
The rebinding kinetics of CO to protoheme (FePPIX) in the presence and absence of a proximal imidazole ligand reveals the magnitude of the rebinding barrier associated with proximal histidine ligation. The ligation states of the heme under different solvent conditions are also investigated using both equilibrium and transient spectroscopy. In the absence of imidazole, a weak ligand (probably water) is bound on the proximal side of the FePPIX-CO adduct. When the heme is encapsulated in micelles of cetyltrimethylammonium bromide (CTAB), photolysis of FePPIX-CO induces a complicated set of proximal ligation changes. In contrast, the use of glycerol-water solutions leads to a simple two-state geminate kinetic response with rapid (10–100 ps) CO recombination and a geminate amplitude that can be controlled by adjusting the solvent viscosity. By comparing the rate of CO rebinding to protoheme in glycerol solution with and without a bound proximal imidazole ligand, we find the enthalpic contribution to the proximal rebinding barrier, Hp, to be 11 ± 2 kJ/mol. Further comparison of the CO rebinding rate of the imidazole bound protoheme with the analogous rate in myoglobin (Mb) leads to a determination of the difference in their distal free energy barriers: ΔGD ≈ 12 ± 1 kJ/mol. Estimates of the entropic contributions, due to the ligand accessible volumes in the distal pocket and the xenon-4 cavity of myoglobin (~3 kJ/mol), then lead to a distal pocket enthalpic barrier of HD ≈ 9 ± 2 kJ/mol. These results agree well with the predictions of a simple model and with previous independent room-temperature measurements (Tian et al. Phys. Rev. Lett. 1992, 68, 408) of the enthalpic MbCO rebinding barrier (18 ± 2 kJ/mol).
Related Articles
Nitrosylation Mechanisms of Mycobacterium tuberculosis and Campylobacter jejuni Truncated Hemoglobins N, O, and P
Ascenzi, Paolo
di Masi, Alessandra
Tundo, Grazia R.
Pesce, Alessandra
Visca, Paolo
Coletta, Massimo
PLoS ONE
Truncated hemoglobins (trHbs) are widely distributed in bacteria and plants and have been found in some unicellular eukaryotes. Phylogenetic analysis based on protein sequences shows that trHbs branch into three groups, designated N (or I), O (or II), and P (or III). Most trHbs are involved in the O2/NO chemistry and/or oxidation/reduction function, permitting the survival of the microorganism in the host. Here, a detailed comparative analysis of kinetics and/or thermodynamics of (i) ferrous Mycobacterium tubertulosis trHbs N and O (Mt-trHbN and Mt-trHbO, respectively), and Campylobacter jejuni trHb (Cj-trHbP) nitrosylation, (ii) nitrite-mediated nitrosylation of ferrous Mt-trHbN, Mt-trHbO, and Cj-trHbP, and (iii) NO-based reductive nitrosylation of ferric Mt-trHbN, Mt-trHbO, and Cj-trHbP is reported. Ferrous and ferric Mt-trHbN and Cj-trHbP display a very high reactivity towards NO; however, the conversion of nitrite to NO is facilitated primarily by ferrous Mt-trHbN. Values of kinetic and/or thermodynamic parameters reflect specific trHb structural features, such as the ligand diffusion pathways to/from the heme, the heme distal pocket structure and polarity, and the ligand stabilization mechanisms. In particular, the high reactivity of Mt-trHbN and Cj-trHbP reflects the great ligand accessibility to the heme center by two protein matrix tunnels and the E7-path, respectively, and the penta-coordination of the heme-Fe atom. In contrast, the heme-Fe atom of Mt-trHbO the ligand accessibility to the heme center of Mt-trHbO needs large conformational readjustments, thus limiting the heme-based reactivity. These results agree with different roles of Mt-trHbN, Mt-trHbO, and Cj-trHbP in vivo.
Related Articles
Peroxidase Activity and Involvement in the Oxidative Stress Response of Roseobacter denitrificans Truncated Hemoglobin
Wang, Yaya
Barbeau, Xavier
Bilimoria, Astha
Lagüe, Patrick
Couture, Manon
Tang, Joseph Kuo-Hsiang
PLoS ONE
Roseobacter denitrificans is a member of the widespread marine Roseobacter genus. We report the first characterization of a truncated hemoglobin from R. denitrificans (Rd. trHb) that was purified in the heme-bound form from heterologous expression of the protein in Escherichia coli. Rd. trHb exhibits predominantly alpha-helical secondary structure and absorbs light at 412, 538 and 572 nm. The phylogenetic classification suggests that Rd. trHb falls into group II trHbs, whereas sequence alignments indicate that it shares certain important heme pocket residues with group I trHbs in addition to those of group II trHbs. The resonance Raman spectra indicate that the isolated Rd. trHb contains a ferric heme that is mostly 6-coordinate low-spin and that the heme of the ferrous form displays a mixture of 5- and 6-coordinate states. Two Fe-His stretching modes were detected, notably one at 248 cm-1, which has been reported in peroxidases and some flavohemoglobins that contain an Fe-His-Asp (or Glu) catalytic triad, but was never reported before in a trHb. We show that Rd. trHb exhibits a significant peroxidase activity with a (kcat/Km) value three orders of magnitude higher than that of bovine Hb and only one order lower than that of horseradish peroxidase. This enzymatic activity is pH-dependent with a pKa value ~6.8. Homology modeling suggests that residues known to be important for interactions with heme-bound ligands in group II trHbs from Mycobacterium tuberculosis and Bacillus subtilis are pointing toward to heme in Rd. trHb. Genomic organization and gene expression profiles imply possible functions for detoxification of reactive oxygen and nitrogen species in vivo. Altogether, Rd. trHb exhibits some distinctive features and appears equipped to help the bacterium to cope with reactive oxygen/nitrogen species and/or to operate redox biochemistry.
Related Articles
Real-time tracking of CO migration and binding in the α and β subunits of human hemoglobin via 150-ps time-resolved Laue crystallography
Schotte, Friedrich
Cho, Hyun Sun
Soman, Jayashree
Wulff, Michael
Olson, John S.
Anfinrud, Philip A.
Chemical physics
We have developed the method of picosecond Laue crystallography and used this capability to probe ligand dynamics in tetrameric R-state hemoglobin (Hb). Time-resolved, 2 Å-resolution electron density maps of photolyzed HbCO reveal the time-dependent population of CO in the binding (A) and primary docking (B) sites of both α and β subunits from 100 ps to 10 μs. The proximity of the B site in the β subunit is about 0.25 Å closer to its A binding site, and its kBA rebinding rate (~300 μs−1) is six times faster, suggesting distal control of the rebinding dynamics. Geminate rebinding in the β subunit exhibits both prompt and delayed geminate phases. We developed a microscopic model to quantitatively explain the observed kinetics, with three states for the α subunit and four states for the β subunit. This model provides a consistent framework for interpreting rebinding kinetics reported in prior studies of both HbCO and HbO2.
Hemoglobin; Geminate rebinding; Ligand migration; Time-resolved Laue crystallography
Related Articles
Responses of Mycobacterium tuberculosis Hemoglobin Promoters to In Vitro and In Vivo Growth Conditions▿
Pawaria, Sudesh
Lama, Amrita
Raje, Manoj
Dikshit, Kanak L.
Applied and Environmental Microbiology
The success of Mycobacterium tuberculosis as one of the dreaded human pathogens lies in its ability to utilize different defense mechanisms in response to the varied environmental challenges during the course of its intracellular infection, latency, and reactivation cycle. Truncated hemoglobins trHbN and trHbO are thought to play pivotal roles in the cellular metabolism of this organism during stress and hypoxia. To delineate the genetic regulation of the M. tuberculosis hemoglobins, transcriptional fusions of the promoters of the glbN and glbO genes with green fluorescent protein were constructed, and their responses were monitored in Mycobacterium smegmatis and M. tuberculosis H37Ra exposed to environmental stresses in vitro and in M. tuberculosis H37Ra after in vivo growth inside macrophages. The glbN promoter activity increased substantially during stationary phase and was nearly 3- to 3.5-fold higher than the activity of the glbO promoter, which remained more or less constant during different growth phases in M. smegmatis, as well as in M. tuberculosis H37Ra. In both mycobacterial hosts, the glbN promoter activity was induced 1.5- to 2-fold by the general nitrosative stress inducer, nitrite, as well as the NO releaser, sodium nitroprusside (SNP). The glbO promoter was more responsive to nitrite than to SNP, although the overall increase in its activity was much less than that of the glbN promoter. Additionally, the glbN promoter remained insensitive to the oxidative stress generated by H2O2, but the glbO promoter activity increased nearly 1.5-fold under similar conditions, suggesting that the trHb gene promoters are regulated differently under nitrosative and oxidative stress conditions. In contrast, transition metal-induced hypoxia enhanced the activity of both the glbN and glbO promoters at all growth phases; the glbO promoter was induced ∼2.3-fold, which was found to be the highest value for this promoter under all the conditions evaluated. Addition of iron along with nickel reversed the induction in both cases. Interestingly, a concentration-dependent decrease in the activity of both trHb gene promoters was observed when the levels of iron in the growth media were depleted by addition of an iron chelator. These results suggested that an iron/heme-containing oxygen sensor is involved in the modulation of the trHb gene promoter activities directly or indirectly in conjunction with other cellular factors. The modes of promoter regulation under different physiological conditions were found to be similar for the trHbs in both M. smegmatis and M. tuberculosis H37Ra, indicating that the promoters might be regulated by components that are common to the two systems. Confocal microscopy of THP-1 macrophages infected with M. tuberculosis carrying the trHb gene promoter fusions showed that there was a significant level of promoter activity during intracellular growth in macrophages. Time course evaluation of the promoter activity after various times up to 48 h by fluorescence-activated cell sorting analysis of the intracellular M. tuberculosis cells indicated that the glbN promoter was active at all time points assessed, whereas the activity of the glbO promoter remained at a steady-state level up to 24 h postinfection and increased ∼2-fold after 48 h of infection. Thus, the overall regulation pattern of the M. tuberculosis trHb gene promoters correlates not only with the stresses that the tubercle bacillus is likely to encounter once it is in the macrophage environment but also with our current knowledge of their functions. The in vivo studies that demonstrated for the first time expression of trHbs during macrophage infection of M. tuberculosis strongly indicate that the hemoglobins are required, and thus important, during the intracellular phase of the bacterial cycle. The present study of transcriptional regulation of M. tuberculosis hemoglobins in vitro under various stress conditions and in vivo after macrophage infection supports the hypothesis that biosynthesis of both trHbs (trHbN and trHbO) in the native host is regulated via the environmental signals that the tubercle bacillus receives during macrophage infection and growth in its human host.
Related Articles
Vibrational Coherence Spectroscopy of the Heme Domain in the CO-Sensing Transcriptional Activator CooA
Karunakaran, Venugopal
Benabbas, Abdelkrim
Youn, Hwan
Champion, Paul M.
Journal of the American Chemical Society
Femtosecond vibrational coherence spectroscopy was used to investigate the low frequency vibrational dynamics of the heme in the carbon monoxide oxidation activator protein (CooA) from the thermophilic anaerobic bacterium Carboxydothermus hydrogenoformans (Ch-CooA). Low frequency vibrational modes are important because they are excited by the ambient thermal bath (kBT = 200 cm-1) and participate in thermally activated barrier crossing events. However, such modes are nearly impossible to detect in the aqueous phase using traditional spectroscopic methods. Here we present the low frequency coherence spectra of the ferric, ferrous, and CO-bound forms of Ch-CooA in order to compare the protein-induced heme distortions in its active and inactive states. Distortions take place predominantly along the coordinates of low-frequency modes because of their weak force constants and such distortions are reflected in the intensity of the vibrational coherence signals. A strong mode near ~90 cm-1 in the ferrous form of Ch-CooA is suggested to contain a large component of heme ruffling, consistent with the imidazole bound ferrous heme crystal structure, which shows a significant protein-induced heme distortion along this coordinate. A mode observed at ~228 cm-1 in the six-coordinate ferrous state is proposed to be the ν(Fe-His) stretching vibration. The observation of the Fe-His mode indicates that photolysis of the N-terminal α-amino axial ligand takes place. This is followed by a rapid (~8.5 ps) transient absorption recovery, analogous to methionine rebinding in photolyzed ferrous cytochrome c. We have also studied CO photolysis in CooA, which revealed very strong photoproduct state coherent oscillations. The observation of heme-CO photoproduct oscillations is unusual because most other heme systems have CO rebinding kinetics that are too slow to make the measurement possible. The low frequency coherence spectrum of the CO-bound form of Ch-CooA shows a strong vibration at ~230 cm-1 that is broadened and up-shifted compared to the ν(Fe-His) of Rr-CooA (216 cm-1). We propose that the stronger Fe-His bond is related to the enhanced thermal stability of Ch-CooA and that there is a smaller (time dependent) tilt of the histidine ring with respect to the heme plane in Ch-CooA. The appearance of strong modes at ~48 cm-1 in both the ferrous and CO-bound forms of Ch-CooA is consistent with coupling of the heme doming distortion to the photolysis reaction in both samples. Upon CO binding and protein activation, a heme mode near 112 ± 5 cm-1 disappears, probably indicating a decreased heme saddling distortion. This reflects changes in the heme environment and geometry that must be associated with the conformational transition activating the DNA-binding domain. Protein-specific DNA binding to the CO-bound form of Ch-CooA was also investigated and, although the CO rebinding kinetics are significantly perturbed, there are negligible changes in the low-frequency vibrational spectrum of the heme.
Ch-CooA; DNA-Protein interaction; low frequency modes; heme distortion; Fe-His stretching vibration
Related Articles
Modulating Distal Cavities in the α and β Subunits of Human HbA Reveals the Primary Ligand Migration Pathway†
Birukou, Ivan
Maillett, David H.
Birukova, Anastasiya
Olson, John S.
The free volume in the active site of human HbA plays a crucial role in governing the bimolecular rates of O2, CO, and NO binding, the fraction of geminate ligand recombination, and the rate of NO dioxygenation by the oxygenated complex. We have decreased the size of the distal pocket by mutating Leu(B10), Val(E11) and Leu(G8) to Phe and Trp and of other more internal cavities by filling them with Xe at high gas pressures. Increasing the size of the B10 side chain reduces bimolecular rates of ligand binding nearly 5,000-fold and inhibits CO geminate recombination due to both reduction of the capture volume in the distal pocket and direct steric hindrance of Fe-ligand bond formation. Phe and Trp(E11) mutations also cause a decrease in distal pocket volume but, at the same time, increase access to the Fe atom due to the loss of the γ2 CH3 group of the native Val(E11) side chain. The net result of these E11 substitutions is a dramatic increase in geminate recombination because dissociated CO is sequestered close to the Fe atom and can rapidly rebind without steric resistance. However, the bimolecular rate constants for ligand binding to the Phe and Trp(E11) mutants are decreased 5–30-fold, due to a smaller capture volume. Geminate and bimolecular kinetic parameters for Phe and Trp(G8) mutants are similar to those for the native HbA subunits because the aromatic rings at this position cause little change in distal pocket volume and because ligands do not move past this position into the globin interior of wild-type HbA subunits. The latter conclusion is verified by the observation that Xe binding to the α and β Hb subunits has little effect on either geminate or bimolecular ligand rebinding. All of these experimental results argue strongly against alternative ligand migration pathways that involve movements through the protein interior in HbA. Instead, ligands appear to enter through the His(E7) gate and are captured directly in the distal cavity.
hemoglobin; myoglobin; xenon binding sites; geminate recombination; flash photolysis; ligand migration pathways; non-covalent binding site
Related Articles
Role of Copper Ion in Regulating Ligand Binding in a Myoglobin-Based Cytochrome c Oxidase Model
Lu, Changyuan
Zhao, Xuan
Lu, Yi
Rousseau, Denis L.
Yeh, Syun-Ru
Journal of the American Chemical Society
Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a3) and a copper atom (CuB) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named CuBMb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in CuBMb was investigated. Resonance Raman studies show that the presence of copper in CO-bound CuBMb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu+; it subsequently transfers from Cu+ to Fe2+ in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu+ and the slow intramolecular CO transfer rate between Cu+ and Fe2+ in the CuBMb/Cu+ complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound CuBMb/Cu+ complex, the photolyzed ligand transiently binds to Cu+ and subsequently rebinds to Fe2+, accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the CuBMb/Cu+ complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.
Related Articles
The Stretching Frequencies of Bound Alkyl Isocyanides Indicate Two Distinct Ligand Orientations within the Distal Pocket of Myoglobin†
Blouin, George C.
Olson, John S.
The FTIR spectra for alkyl isocyanides (CNRs) change from a single νCN band centered at ~2175 cm−1 to two peaks at ~2075 and ~2125 cm−1 upon binding to sperm whale myoglobin (Mb). The low and high frequency peaks have been assigned to in and out conformations, respectively. In the in conformation, the ligand is pointing toward the protein interior, and the distal His64(E7) is in a closed position, donates a H-bond to the bound isocyano group, enhances back bonding, and lowers the C-N bond order. In the out conformation, the ligand side chain points toward solvent through a channel opened by outward rotation of His64. Loss of positive polarity near the binding site causes an increase in C-N bond order. Support for this interpretation is three-fold: (1) similar shifts to lower frequency occur for MbCO complexes when H-bond donation from His64(E7) occurs; (2) only one peak at ~2125 cm−1, indicative of an apolar environment, is observed for CNRs bound to H64A or H64L Mb mutants or to chelated protoheme in soap micelles; and (3) the fraction of in conformation based on FTIR spectra correlates strongly with the fraction of geminate recombination after nanosecond laser photolysis. The in alkyl side chain conformation causes the photodissociated ligand to be "stuck" in the distal pocket, promoting internal rebinding, whereas the out conformation inhibits geminate recombination because part of the ligand is already in an open E7 channel, poised for rapid escape.
myoglobin; alkyl isocyanide; isonitrile; model heme; soap micelle; FTIR; infrared; vibrational spectroscopy; Fermi resonance; geminate recombination; iron coordination
Related Articles
Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy
Rappaport, Fabrice
Zhang, Jie
Vos, Marten H.
Gennis, Robert B.
Borisov, Vitaliy B.
Biochimica et biophysica acta
Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b558, b595, and d. The role of heme b595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully-reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully-reduced enzyme. The difference demonstrates that in the fully-reduced enzyme photolysis of CO from heme d perturbs ferrous heme b595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully-reduced enzyme monoexponentially with τ ~12 µs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ~14 ns, 14 µs, and 280 µs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully-reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14 µs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ~4% of the enzyme population. The final, 280 µs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b595, and not that of heme b558, controls the pathway(s) by which CO migrates between heme d and the medium.
respiration; chlorin; cytochrome; ligand binding; gas molecule; photobiology
Related Articles
Direct Observation of Cooperative Protein Structural Dynamics of Homodimeric Hemoglobin from 100 ps to 10 ms with Pump–Probe X-ray Solution Scattering
Kim, Kyung Hwan
Muniyappan, Srinivasan
Oang, Key Young
Kim, Jong Goo
Nozawa, Shunsuke
Sato, Tokushi
Koshihara, Shin-ya
Henning, Robert
Kosheleva, Irina
Ki, Hosung
Kim, Youngmin
Kim, Tae Wu
Kim, Jeongho
Adachi, Shin-ichi
Ihee, Hyotcherl
Journal of the American Chemical Society
Proteins serve as molecular machines in performing their biological functions, but the detailed structural transitions are difficult to observe in their native aqueous environments in real time. For example, despite extensive studies, the solution-phase structures of the intermediates along the allosteric pathways for the transitions between the relaxed (R) and tense (T) forms have been elusive. In this work, we employed picosecond X-ray solution scattering and novel structural analysis to track the details of the structural dynamics of wild-type homodimeric hemoglobin (HbI) from the clam Scapharca inaequivalvis and its F97Y mutant over a wide time range from 100 ps to 56.2 ms. From kinetic analysis of the measured time-resolved X-ray solution scattering data, we identified three structurally distinct intermediates (I1, I2, and I3) and their kinetic pathways common for both the wild type and the mutant. The data revealed that the singly liganded and unliganded forms of each intermediate share the same structure, providing direct evidence that the ligand photolysis of only a single subunit induces the same structural change as the complete photolysis of both subunits does. In addition, by applying novel structural analysis to the scattering data, we elucidated the detailed structural changes in the protein, including changes in the heme–heme distance, the quaternary rotation angle of subunits, and interfacial water gain/loss. The earliest, R-like I1 intermediate is generated within 100 ps and transforms to the R-like I2 intermediate with a time constant of 3.2 ± 0.2 ns. Subsequently, the late, T-like I3 intermediate is formed via subunit rotation, a decrease in the heme–heme distance, and substantial gain of interfacial water and exhibits ligation-dependent formation kinetics with time constants of 730 ± 120 ns for the fully photolyzed form and 5.6 ± 0.8 μs for the partially photolyzed form. For the mutant, the overall kinetics are accelerated, and the formation of the T-like I3 intermediate involves interfacial water loss (instead of water entry) and lacks the contraction of the heme–heme distance, thus underscoring the dramatic effect of the F97Y mutation. The ability to keep track of the detailed movements of the protein in aqueous solution in real time provides new insights into the protein structural dynamics.
Related Articles
Ultrafast dynamics of diatomic ligand binding to nitrophorin 4
Benabbas, Abdelkrim
Ye, Xiong
Kubo, Minoru
Zhang, Zhenyu
Maes, Estelle M.
Montfort, William R.
Champion, Paul M.
Journal of the American Chemical Society
Nitrophorin 4 (NP4) is a heme protein that stores and delivers nitric oxide (NO) through pH sensitive conformational change. This protein uses the ferric state of a highly ruffled heme to bind NO tightly at low pH and release it at high pH. In this work, the rebinding kinetics of NO and CO to NP4 are investigated as a function of iron oxidation state and the acidity of the environment. The geminate recombination process of NO to ferrous NP4 at both pH 5 and pH 7 is dominated by a single ~7 ps kinetic phase that we attribute to the rebinding of NO directly from the distal pocket. The lack of pH dependence explains in part why NP4 cannot use the ferrous state to fulfill its function. The kinetic response of ferric NP4NO shows two distinct phases. The relative geminate amplitude of the slower phase increases dramatically as the pH is raised from 5 to 8. We assign the fast phase of NO rebinding to a conformation of the ferric protein with a closed hydrophobic pocket. The slow phase is assigned to the protein in an open conformation with a more hydrophilic heme pocket environment. Analysis of the ultrafast kinetics finds the equilibrium off-rate of NO to be proportional to the open state population as well as the pH-dependent amplitude of escape from the open pocket. When both factors are considered, the off-rate increases by more than an order of magnitude as the pH changes from 5 to 8. The recombination of CO to ferrous NP4 is observed to have a large non-exponential geminate amplitude with rebinding timescales of ~10−11–10−9 s at pH 5 and ~10−10–10−8 s at pH 7. The non-exponential CO rebinding kinetics at both pH 5 and pH 7 are accounted for using a simple model that has proven effective for understanding CO binding in a variety of other heme systems.
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An Engineered Heme-Copper Center in Myoglobin: CO Migration and Binding
Nienhaus, Karin
Olson, John S.
Nienhaus, G. Ulrich
Biochimica et biophysica acta
We have investigated CO migration and binding in CuBMb, a copper-binding myoglobin double mutant (L29H-F43H), by using Fourier transform infrared spectroscopy and flash photolysis over a wide temperature range. This mutant was originally engineered with the aim to mimic the catalytic site of heme-copper oxidases. Comparison of the wild-type protein Mb and CuBMb shows that the copper ion in the distal pocket gives rise to significant effects on ligand binding to the heme iron. In Mb and copper-free CuBMb, primary and secondary ligand docking sites are accessible upon photodissociation. In copper-bound CuBMb, ligands do not migrate to secondary docking sites but rather coordinate to the copper ion. Ligands entering the heme pocket from the outside normally would not be captured efficiently by the tight distal pocket housing the two additional large imidazole rings. Binding at the Cu ion, however, ensures efficient trapping in CuBMb. The Cu ion also restricts the motions of the His64 side chain, which is the entry/exit door for ligand movement into the active site, and this restriction results in enhanced geminate and slow bimolecular CO rebinding. These results support current mechanistic views of ligand binding in hemoglobins and the role of the CuB in the active of heme-copper oxidases.
Fourier transform infrared spectroscopy; temperature derivative spectroscopy; heme protein; ligand binding; ligand migration; photolysis difference spectroscopy
Related Articles
Temperature dependent studies of NO recombination to heme and heme proteins
Ionascu, Dan
Gruia, Flaviu
Ye, Xiong
Yu, Anchi
Rosca, Florin
Beck, Chris
Demidov, Andrey
Olson, John S.
Champion, Paul M.
Journal of the American Chemical Society
The rebinding kinetics of NO to the heme iron of myoglobin (Mb) is investigated as a function of temperature. Below 200K, the transition state enthalpy barrier associated with the fastest (~10ps) recombination phase is found to be zero, while a slower geminate phase (~200ps) reveals a small enthalpic barrier (~ 3 ± 1 kJ/mol). Both of the kinetic rates slow down slightly in the myoglobin (Mb) samples above 200K, suggesting that a small amount of protein relaxation takes place above the solvent glass transition. When the temperature dependence of the NO recombination in Mb is studied under conditions where the distal pocket is mutated (e.g., V68W), the rebinding kinetics lack the slow phase. This is consistent with a mechanism where the slower (~200ps) kinetic phase involves transitions of the NO ligand into the distal heme pocket from a more distant site (e.g., in or near the Xe4 cavity). Comparison of the temperature dependent NO rebinding kinetics of native Mb with that of the bare heme (PPIX) in glycerol reveals that the fast (enthalpically barrierless) NO rebinding process observed below 200K is independent of the presence or absence of the proximal histidine ligand. In contrast, the slowing of the kinetic rates above 200K in MbNO disappears in the absence of the protein. Generally, the data indicate that, in contrast to CO, the NO ligand binds to the heme iron through a "harpoon" mechanism where the heme iron out-of-plane conformation presents a negligible enthalpic barrier to NO rebinding. These observations strongly support a previous analysis (J. Am. Chem. Soc. 1988, 110, 6656) that primarily attributes the low temperature stretched exponential rebinding of MbCO to a quenched distribution of heme geometries. A simple model is presented for MbNO rebinding that explains a variety of experiments, including the dependence of the kinetic amplitudes on the pump photon energy.
Related Articles
Nanosecond Time-Resolved Polarization Spectroscopies: Tools for Probing Protein Reaction Mechanisms
Chen, Eefei
Goldbeck, Robert A.
Kliger, David S.
Methods (San Diego, Calif.)
Polarization methods, introduced in the 1800's, offered one of the earliest ways to examine protein structure. Since then, many other structure-sensitive probes have been developed, but circular dichroism (CD) remains a powerful technique because of its versatility and the specificity of protein structural information that can be explored. With improvements in time-resolution, from millisecond to picosecond CD measurements, it has proven to be an important tool for studying the mechanism of folding and function in many biomolecules. For example, nanosecond time-resolved CD (TRCD) studies of the sub-microsecond events of reduced cytochrome c folding have provided direct experimental evidence of kinetic heterogeneity, which is an inherent property of the diffusional nature of early folding dynamics on the energy landscape. In addition, TRCD has been applied to the study of many biochemical processes, such as ligand rebinding in hemoglobin and myoglobin and signaling state formation in photoactive yellow protein and prototropin 1 LOV2. The basic approach to TRCD has also been extended to include a repertoire of nanosecond polarization spectroscopies: optical rotatory dispersion (ORD), magnetic CD and ORD, and linear dichroism. This article will discuss the details of the polarization methods used in this laboratory, as well as the coupling of timeresolved ORD with the temperature-jump trigger so that protein folding can be studied in a larger number of proteins.
Time-resolved; Circular dichroism; Temperature-jump; Optical rotatory dispersion; Magnetic circular dichroism; Polarization spectroscopy
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Evolution, Three-Dimensional Model and Localization of Truncated Hemoglobin PttTrHb of Hybrid Aspen
Dumont, Estelle
Jokipii-Lukkari, Soile
Parkash, Vimal
Vuosku, Jaana
Sundström, Robin
Nymalm, Yvonne
Sutela, Suvi
Taskinen, Katariina
Kallio, Pauli T.
Salminen, Tiina A.
Häggman, Hely
PLoS ONE
Thus far, research on plant hemoglobins (Hbs) has mainly concentrated on symbiotic and non-symbiotic Hbs, and information on truncated Hbs (TrHbs) is scarce. The aim of this study was to examine the origin, structure and localization of the truncated Hb (PttTrHb) of hybrid aspen (Populus tremula L. × tremuloides Michx.), the model system of tree biology. Additionally, we studied the PttTrHb expression in relation to non-symbiotic class1 Hb gene (PttHb1) using RNAi-silenced hybrid aspen lines. Both the phylogenetic analysis and the three-dimensional (3D) model of PttTrHb supported the view that plant TrHbs evolved vertically from a bacterial TrHb. The 3D model suggested that PttTrHb adopts a 2-on-2 sandwich of α-helices and has a Bacillus subtilis -like ligand-binding pocket in which E11Gln and B10Tyr form hydrogen bonds to a ligand. However, due to differences in tunnel cavity and gate residue (E7Ala), it might not show similar ligand-binding kinetics as in Bs-HbO (E7Thr). The immunolocalization showed that PttTrHb protein was present in roots, stems as well as leaves of in vitro -grown hybrid aspens. In mature organs, PttTrHb was predominantly found in the vascular bundles and specifically at the site of lateral root formation, overlapping consistently with areas of nitric oxide (NO) production in plants. Furthermore, the NO donor sodium nitroprusside treatment increased the amount of PttTrHb in stems. The observed PttTrHb localization suggests that PttTrHb plays a role in the NO metabolism.
Related Articles
Laser Flash Photolysis Generation of High-Valent Transition Metal-Oxo Species: Insights from Kinetic Studies in Real Time
Zhang, Rui
Newcomb, Martin
Accounts of chemical research
High-valent transition metal-oxo species are active oxidizing species in many metal-catalyzed oxidation reactions in both Nature and the laboratory. In homogeneous catalytic oxidations, a transition metal catalyst is oxidized to a metal-oxo species by a sacrificial oxidant, and the activated transition metal-oxo intermediate oxidizes substrates. Mechanistic studies of these oxidizing species can provide insights for understanding commercially important catalytic oxidations and the oxidants in cytochrome P450 enzymes. In many cases, however, the transition metal oxidants are so reactive that they do not accumulate to detectable levels in mixing experiments, which have millisecond mixing times, and successful generation and direct spectroscopic characterization of these highly reactive transients remain a considerable challenge. Our strategy for understanding homogeneous catalysis intermediates employs photochemical generation of the transients with spectroscopic detection on time-scales as short as nanoseconds and direct kinetic studies of their reactions with substrates by laser flash photolysis (LFP) methods. This Account describes studies of high-valent manganese- and iron-oxo intermediates. Irradiation of porphyrin-manganese(III) nitrates and chlorates or corrole-manganese(IV) chlorates resulted in homolytic cleavage of the O-X bonds in the ligands, whereas irradiation of porphyrin-manganese(III) perchlorates resulted in heterolytic cleavage of O-Cl bonds to give porphyrin-manganese(V)-oxo cations. Similar reactions of corrole- and porphyrin-iron(IV) complexes gave highly reactive transients that were tentatively identified as macrocyclic ligand-iron(V)-oxo species. Kinetic studies demonstrated high reactivity of the manganese(V)-oxo species, and even higher reactivities of the putative iron(V)-oxo transients. For example, second-order rate constants for oxidations of cis-cyclooctene at room temperature were 6 × 103 M−1 s−1 for a corrole-iron(V)-oxo species and 1.6 × 106 M−1 s−1 for the putative tetramesitylporphyrin-iron(V)-oxo perchlorate species. The latter rate constant is 25,000 times larger than that for oxidation of cis-cyclooctene by iron(IV)-oxo perchlorate tetramesitylporphyrin radical cation, which is the thermodynamically favored electronic isomer of the putative iron(V)-oxo species. The LFP-determined rate constants can be used to implicate the transient oxidants in catalytic reactions under turnover conditions where high-valent species are not observable. Similarly, the observed reactivities of the putative porphyrin-iron(V)-oxo species might explain the unusually high reactivity of oxidants produced in the cytochrome P450 enzymes, heme-thiolate enzymes that are capable of oxidizing unactivated carbon-hydrogen bonds in substrates so rapidly that iron-oxo intermediates have not been detected under physiological conditions.
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Investigations of Low Frequency Vibrational Dynamics and Ligand Binding Kinetics of Cystathionine β-synthase
Karunakaran, Venugopal
Benabbas, Abdelkrim
Sun, Yuhan
Zhang, Zhenyu
Singh, Sangita
Banerjee, Ruma
Champion, Paul M.
The journal of physical chemistry. B
Vibrational coherence spectroscopy is used to study the low frequency dynamics of the truncated dimer of human cystathionine β-synthase (CBS). CBS is a pyridoxal-5′-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form cystathionine. A strong correlation between the "detuned" coherence spectrum (which probes higher frequencies) and the Raman spectrum is demonstrated and a rich pattern of modes below 200 cm−1 is revealed. Normal coordinate structural decomposition (NSD) of the ferric CBS crystal structure predicts the enhancement of normal modes with significant heme "doming", "ruffling", and "saddling" content and they are observed in the coherence spectra near ~40 cm−1, ~60 cm−1, and ~90 cm−1. When pH is varied, the relative intensities and frequencies of the low-frequency heme modes indicate the presence of a unique protein-induced heme structural perturbation near pH 7 that differs from what is observed at higher or lower pH. For ferric CBS, we observe a new mode near ~25 cm−1, possibly involving the response of the protein, which exhibits a phase jump of ~π for excitation on the blue and red side of the Soret band maximum. The low frequency vibrational coherence spectrum of ferrous CBS is also presented, along with our efforts to probe its NO-bound complex. The CBS-CO geminate rebinding kinetics are similar to the CO-bound form of the gene activator protein CooA, but with the appearance of a significant additional kinetic inhomogeneity. Analysis of this inhomogeneity suggests that it arises from the two subunits of CBS and leads to a factor of ~20 for the ratio of the average CO geminate rebinding rates of the two subunits.
femtosecond pump-probe; vibrational coherence spectroscopy; cystathionine β-synthase; low frequency modes; geminate rebinding; heme proteins
Related Articles
Photolysis of Adenosylcobalamin and Radical Pair Recombination in Ethanolamine Ammonia-Lyase Probed on the Micro- to Millisecond Time Scale by using Time-Resolved Optical Absorption Spectroscopy†
Robertson, Wesley D.
Warncke, Kurt
The quantum yield and kinetics of decay of cob(II)alamin formed by pulsed-laser photolysis of adenosylcobalamin (AdoCbl) in coenzyme B12 (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied on the 10-7 - 10-1 s time scale at 295 K by using transient ultraviolet-visible absorption spectroscopy. The aim is to probe the mechanism of formation and stabilization of the cob(II)alamin-5′-deoxyadenosyl radical pair, which is a key intermediate in EAL catalysis, and the influence of substrate binding on this process. Substrate binding is required for cobalt-carbon bond cleavage in the native system. Photolysis of AdoCbl in EAL leads to a quantum yield at 10-7 s for cob(II)alamin of 0.08 ±0.01, which is 3-fold less than for AdoCbl in aqueous solution (0.23 ±0.01). The protein binding site therefore suppresses photoproduct radical pair formation. Three photoproduct states, Pf, Ps, and Pc, are identified in holo-EAL by the different cob(II)alamin decay kinetics (subscripts denote fast, slow, and constant, respectively). These states have the following first-order decay rate constants and quantum yields: Pf (2.2×103 s-1; 0.02), Ps (4.2×102 s-1; 0.01), and Pc (constant amplitude, no recombination; 0.05). Binding of the substrate analog, (S)-1-amino-2-propanol, to EAL eliminates the Pf state, and lowers the quantum yield of Pc (0.03) relative to Ps (0.01), but does not significantly change the quantum yield or decay rate constant of Ps, relative to holo-EAL. The substrate analog thus influences the quantum yield at 10-7 s by changing the cage escape rate from the geminate cob(II)alamin-5′-deoxyadenosyl radical pair state. However, the predicted substrate analog binding-induced increase in the quantum yield is not observed. It is proposed that the substrate analog does not induce the radical pair stabilizing changes in the protein that are characteristic of true substrates.
Related Articles
Characterization of the truncated hemoglobin THB1 from protein extracts of Chlamydomonas reinhardtii
Johnson, Eric A.
Lecomte, Juliette T.J.
Truncated hemoglobins (TrHbs) belong to the hemoglobin superfamily, but unlike their distant vertebrate relatives, little is known about their principal physiologic functions. Several TrHbs have been studied in vitro using engineered recombinant peptides. These efforts have resulted in a wealth of knowledge about the chemical properties of TrHbs and have generated interesting functional leads. However, questions persist as to how closely these engineered proteins mimic their counterparts within the native cell. In this report, we examined THB1, one of several TrHbs from the model organism Chlamydomonas reinhardtii. The recombinant THB1 (rTHB1) has favorable solubility and stability properties and is an excellent candidate for in vitro characterization. Linking rTHB1 to the in vivo protein is a critical step in understanding the physiologic function of this protein. Using a simplified three-step purification protocol, 3.5-L batches of algal culture were processed to isolate 50–60 μL fractions enriched in THB1. These fractions of C. reinhardtii proteins were then subjected to physical examination. Using gel mobility, optical absorbance and immunoreactivity, THB1 was identified in these enriched fractions and its presence correlated with that of a heme molecule. Mass spectrometry confirmed this cofactor to be a type b heme and revealed that the native protein contains a co-translational modification consistent with amino-terminal acetylation following initial methionine cleavage.
Hemoglobins; oxygen binding; THB1; Chlamydomonas reinhardtii
Related Articles
Kinetic Spectroscopy of Heme Hydration and Ligand Binding in Myoglobin and Isolated Hemoglobin Chains: An Optical Window into Heme Pocket Water Dynamics
Esquerra, Raymond M.
López-Peña, Ignacio
Tipgunlakant, Pooncharas
Birukou, Ivan
Nguyen, Rosa L.
Soman, Jayashree
Olson, John S.
Kliger, David S.
Goldbeck, Robert A.
Physical chemistry chemical physics : PCCP
The entry of a water molecule into the distal heme pocket of pentacoordinate heme proteins such as myoglobin and the α,β chains of hemoglobin can be detected by time-resolved spectroscopy in the heme visible bands after photolysis of the CO complex. Reviewing the evidence from spectrokinetic studies of Mb variants, we find that this optical method measures the occupancy of non(heme)coordinated water in the distal pocket, nw, with high fidelity. This evidence further suggests that perturbation of the kinetic barrier presented by distal pocket water is often the dominant mechanism by which active site mutations affect the bimolecular rate constant for CO binding. Water entry into the heme pockets of isolated hemoglobin subunits was detected by optical methods. Internal hydration is higher in the native α chains than in the β chains, in agreement with previous crystallographic results for the subunits within Hb tetramers. The kinetic parameters obtained from modeling of the water entry and ligand rebinding in Mb mutants and native Hb chains are consistent with an inverse dependence of the bimolecular association rate constant on the water occupancy factor. This correlation suggests that water and ligand mutually exclude one another from the distal pockets of both types of hemoglobin chains and myoglobin..
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Cloning and Heterologous Expression of a β-d-Mannosidase (EC 184.108.40.206)-Encoding Gene from Thermobifida fusca TM51
Béki, Emese
Nagy, István
Vanderleyden, Jos
Jäger, Szilvia
Kiss, László
Fülöp, László
Hornok, László
Kukolya, József
Applied and Environmental Microbiology
Thermobifida fusca TM51, a thermophilic actinomycete isolated from composted horse manure, was found to produce a number of lignocellulose-degrading hydrolases, including endoglucanases, exoglucanases, endoxylanases, β-xylosidases, endomannanases, and β-mannosidases, when grown on cellulose or hemicellulose as carbon sources. β-Mannosidases (EC 184.108.40.206), although contributing to the hydrolysis of hemicellulose fractions, such as galacto-mannans, constitute a lesser-known group of the lytic enzyme systems due to their low representation in the proteins secreted by hemicellulolytic microorganisms. An expression library of T. fusca, prepared in Streptomyces lividans TK24, was screened for β-mannosidase activity to clone genes coding for mannosidases. One positive clone was identified, and a β-mannosidase-encoding gene (manB) was isolated. Sequence analysis of the deduced amino acid sequence of the putative ManB protein revealed substantial similarity to known mannosidases in family 2 of the glycosyl hydrolase enzymes. The calculated molecular mass of the predicted protein was 94 kDa, with an estimated pI of 4.87. S. lividans was used as heterologous expression host for the putative β-mannosidase gene of T. fusca. The purified gene product obtained from the culture filtrate of S. lividans was then subjected to more-detailed biochemical analysis. Temperature and pH optima of the recombinant enzyme were 53°C and 7.17, respectively. Substrate specificity tests revealed that the enzyme exerts only β-d-mannosidase activity. Its kinetic parameters, determined on para-nitrophenyl β-d-mannopyranoside (pNP-βM) substrate were as follows: Km = 180 μM and Vmax = 5.96 μmol min−1 mg−1; the inhibition constant for mannose was Ki = 5.5 mM. Glucono-lacton had no effect on the enzyme activity. A moderate trans-glycosidase activity was also observed when the enzyme was incubated in the presence of pNP-αM and pNP-βM; under these conditions mannosyl groups were transferred by the enzyme from pNP-βM to pNP-αM resulting in the synthesis of small amounts (1 to 2%) of disaccharides.
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A two-parameter kinetic model based on a time-dependent activity coefficient accurately describes enzymatic cellulose digestion
Kostylev, Maxim
Wilson, David
Lignocellulosic biomass is a potential source of renewable, low-carbon-footprint liquid fuels. Biomass recalcitrance and enzyme cost are key challenges associated with the large-scale production of cellulosic fuel. Kinetic modeling of enzymatic cellulose digestion has been complicated by the heterogeneous nature of the substrate and by the fact that a true steady state cannot be attained. We present a two-parameter kinetic model based on the Michaelis-Menten scheme (Michaelis L and Menten ML. (1913) Biochem Z 49:333–369), but with a time-dependent activity coefficient analogous to fractal-like kinetics formulated by Kopelman (Kopelman R. (1988) Science 241:1620–1626). We provide a mathematical derivation and experimental support to show that one of the parameters is a total activity coefficient and the other is an intrinsic constant that reflects the ability of the cellulases to overcome substrate recalcitrance. The model is applicable to individual cellulases and their mixtures at low-to-medium enzyme loads. Using biomass degrading enzymes from a cellulolytic bacterium Thermobifida fusca we show that the model can be used for mechanistic studies of enzymatic cellulose digestion. We also demonstrate that it applies to the crude supernatant of the widely studied cellulolytic fungus Trichoderma reesei and can thus be used to compare cellulases from different organisms. The two parameters may serve a similar role to Vmax, KM, and kcat in classical kinetics. A similar approach may be applicable to other enzymes with heterogeneous substrates and where a steady state is not achievable.
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Investigations of Ferric Heme Cyanide Photodissociation in Myoglobin and Horseradish Peroxidase
Zeng, Weiqiao
Sun, Yuhan
Benabbas, Abdelkrim
Champion, Paul M.
The journal of physical chemistry. B
The photodissociation of cyanide from ferric myoglobin (MbCN) and horseradish peroxidase (HRPCN) has been definitively observed. This has implications for the interpretation of ultrafast IR (Helbing et al. Biophys. J. 2004, 87, 1881–1891) and optical (Gruia et al. Biophys. J. 2008, 94, 2252–2268) studies that had previously suggested the Fe-CN bond was photostable in MbCN. The photolysis of ferric MbCN takes place with a quantum yield of ~75% and the resonance Raman spectrum of the photoproduct observed in steady-state experiments as a function of laser power and sample spinning rate is identical to that of ferric Mb (metMb). The data are quantitatively analyzed using a simple model where cyanide is photodissociated and, although geminate rebinding with a rate kBA ≈ (3.6 ps)−1 is the dominant process, some CN− exits from the distal heme pocket and is replaced by water. Using independently determined values for the CN− association rate, we find that the CN− escape rate from the ferric myoglobin pocket to the solution at 293 K is kout ≈ 1–2 × 107 s−1. This value is very similar to, but slightly larger than, the histidine gated escape rate of CO from Mb (1.1×107 s−1) under the same conditions. The analysis leads to an escape probability kout/(kout+kBA) ~ 10−4, which is unobservable in most time domain kinetic measurements. However, the photolysis is surprisingly easy to detect in Mb using cw resonance Raman measurements. This is due to the anomalously slow CN− bimolecular association rate (170 M−1s−1), which arises from the need for water to exchange at the ferric heme binding site of Mb. In contrast, ferric HRP does not have a heme bound water molecule and its CN− bimolecular association rate is larger by ~103 making the CN− photolysis more difficult to observe.
Myoglobin CN; heme cyanide; photolysis; resonance Raman
Related Articles
Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3)
Su, Lingqia
Chen, Sheng
Yi, Li
Woodard, Ronald W
Chen, Jian
Wu, Jing
Microbial Cell Factories
Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.
T. fusca cutinase was fused with the specific signal peptide of alpha-hemolysin scretion system and expressed in E. coli BL21(DE3). In addition, HlyB and HlyD, strain-specific translocation components of alpha-hemolysin secretion system, were coexpressed to facilitate the enzyme expression. The cultivation of this engineered cell showed that cutinase activity in the culture medium reached 334 U/ml, which is 2.5 times that from type II secretion pathway under the same culture condition. The recombinant cutinase was further purified. Biochemical characterization of purified enzyme, which had an α-hemolysin secretion pathway signal peptide attached, had substrate specificity, pH and temperature profile, as well as application capability in bioscouring similar to that of wild-type cutinase.
In the present study, T. fusca cutinase was successfully secreted to the culture media by α-hemolysin secretion system. This is the first report of cutinase being efficiently secreted by this pathway. Due to the limited cases of successful expression of industrial enzyme by E. coli α-hemolysin secretion system, our study further explored the utilization of this pathway in industrial enzymes.
alpha-hemolysin secretion pathway; cutinase; protein secretion; extracellular production
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Turbo Charging Yield With Options! 0 comments
I want to dive a little further into my first article and talk about the role of options in boosting returns.
Let's go back to my portfolio of 10 stocks: (JPM, GE, MDT, T, F, INTC, MSFT, DE, CVX, and TGT).
For this example, let pretend we've invested $100,000 in the portfolio (it will make it easier to divide or multiply my calculation to apply for one's own investment resources). Splitting the 100K evenly across the 10 stocks -- assuming market prices as of November 26, 2013 -- we should have the following number of stocks per 10K. I've also included dividend yields.
Ticker Price per Share Number of Shares Dividend per Quarter Yield per quarter Annual Yield JPM $57.65 173 $0.38 0.66% 2.64% GE $26.73 374 $0.19 0.71% 2.84% MDT $57.28 174 $0.28 0.49% 1.96% T $35.37 282 $0.45 1.27% 5.09% F $16.94 590 $0.10 0.59% 2.36% INTC $23.75 421 $0.22 0.93% 3.71% MSFT $37.64 265 $0.28 0.74% 2.98% DE $84.68 118 $0.51 0.60% 2.41% CVX $123.74 80 $1.00 0.81% 3.23% TGT $63.76 156 $0.43 0.67% 2.70% Average Yield 0.75% 2.99%
-- Table 1: Portfolio with Dividend Yield. --
Let's use a dividend-reinvestment-program or DRIP to add to my positions quarterly.
Additional Shares Per Position = [(Dividend x Number of Shares) / Share Price]
Ticker New Number of Shares Q1 New Number of Shares Q2 New Number of Shares Q3 New Number of Shares Q4 JPM 174.14 175.29 176.44 177.61 GE 376.66 379.34 382.03 384.75 MDT 174.85 175.71 176.56 177.43 T 285.59 289.22 292.90 296.63 F 593.48 596.99 600.51 604.06 INTC 424.90 428.84 432.81 436.82 MSFT 266.97 268.96 270.96 272.97 DE 118.71 119.43 120.14 120.87 CVX 80.65 81.30 81.96 82.62 TGT 157.05 158.11 159.18 160.25
-- Table 2: DRIP Adjustments per Quarter. --
Assuming no dividend growth for the year, the portfolio DRIP yield is 2.74%.
As I stated in my previous article, I plan on dollar-cost-averaging my investments quarterly (equal-weight additions quarterly). For the example, let's assume we invest $10,000 a quarter. So, we DCA $1000 per position per quarter.
Additional Shares Per Position Per Quarter = ($1000 / Share Price)
Ticker DCA Shares Q1 JPM 17 GE 37 MDT 17 T 28 F 59 INTC 42 MSFT 26 DE 11 CVX 8 TGT 15
-- Table 3: Dollar-cost-averaging additional shares per quarter for a $10,000 quarterly investment. --
So according to my plan, I'm willing to invest the number of shares in the table above per quarter at today's prices. For example, if I had an extra $10,000, I would take $1,000 and buy 42 shares of Intel . I'm willing to buy the shares at today's price, because I think they are a good value.
Unfortunately, I don't have an extra $10,000 at my disposal today. However, I plan to save that much in three months. Is there a way to make use of that "future money?"
This is where options come in...
With options, I can sell someone the right to sell me shares (or put the shares to me) at the price I'm willing to buy at a time I specify and boost my yield in the process. Note: Options are contracts to buy or sell underlying stocks in units of 100. If you're unfamiliar with options, start here...
Let's go back to our example:
I'm willing to invest a $1000 per position per quarter. If I sell cash-secured put options (that are in-the-money as of today) I can earn the following amount per quarter:
Ticker Put Option Value Capital Backup Return on Capital JPM $3.40 $6,000.00 5.67% GE $0.90 $2,700.00 3.33% MDT $1.55 $5,750.00 2.70% T $1.30 $3,600.00 3.61% F $1.24 $1,800.00 6.89% INTC $0.90 $2,400.00 3.75% MSFT $1.54 $3,800.00 4.05% DE $2.75 $8,500.00 3.24% CVX $3.50 $12,500.00 2.80% TGT $1.80 $6,500.00 2.77% TOTAL $1,888.00 $53,550.00
-- Table 4: Selling put contracts to boost return on capital. --
As long as the stock stays below the strike price, the stock is assigned to me at the strike price. If the stock surges ahead of the strike price, no one will sell the shares to me below market value. However, I'll get to pocket the option price. The risk is, if there's a catastrophic event and the stock tanks, I'm forced to buy it at the contract price. However, I already intended to buy the stock at today's price. Why not collect a premium on the price and effectively purchase the stock at a discount? It's like collecting rent on your time.
By selling in-the-money put options in this example, I'm collecting $1888 on $53,550 in capital. You can either have $53k set aside to secure the puts, or you can use a margin account to cover the puts (you're not charged an interest rate unless your account balance is actually below zero).
Of course margin requirements for large-cap stocks are usually less than 100%, so you can leverage more contracts - but remember each expiring in-the-money obligates you to buy 100 shares. That is more than what you planned to dollar-cost average into each quarter (see table 3).
Ideally, the contracts are assigned to you near the strike price. You can then turn around and sell the stock that you were not planning on buying that quarter. The table below shows how many shares per contract would have to be sold if the stock price remains the same.
Ticker # of shares to sell back JPM 83 GE 63 MDT 83 T 72 F 41 INTC 58 MSFT 74 DE 89 CVX 92 TGT 85
-- Table 5: Assume all the contracts are assigned to you and the stock price didn't change. You subtract the number of shares in table 3 by 100 (the number in the contract assigned to you) to get the number of shares to sell after contract assignment. --
Please note that you could also sell-in-the-money covered calls to generate revenue. However, I intend to sell options quarterly - if I sold a call and my shares ended up getting called away from me for a profit - I'd take a short-term gains tax hit. Additionally, it would go against my philosophy of slowly adding to my positions. That said, if a portfolio was large enough that you could sell a few shares - you could setup long-term (greater than 365 day expiration date) strangle options (cash-secure put + covered-call) on your positions. I haven't done the math on that idea yet...
In conclusion:
If you have the discipline to commit to today's prices on the stocks in your portfolio and you're also willing to give up gains above your contract price, then selling in-the-money put contracts might be a way for you to increase your annual income from your capital beyond the yield from dividends. Using my example above you can earn about $2000 extra per quarter than you would from dividends alone. This approximately adds up to an additional 8% per year on top of dividends. Results may fall short of your expectations if the market drops more than what you collect on option premiums, or if the market soars past your strike prices (it's unlikely that you will be correct with your option calls 100% of the time). You can also apply this strategy to riskier stocks like small-caps and momentum stocks. It's not my style, but you'll earn a much higher premium for the added volatility risk.
Disclosure: I am long INTC, MSFT, F, TGT, JPM, MDT, T, GE, CVX, DE.Back To Omar Farooq's Instablog HomePage »
Instablogs are blogs which are instantly set up and networked within the Seeking Alpha community. Instablog posts are not selected, edited or screened by Seeking Alpha editors, in contrast to contributors' articles.Full index of posts »
Want To Find Alpha In This Market? Invest Abroad http://seekingalpha.com/a/1cw2v Jun 23, 2014
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- Want To Find Alpha In This Market? Invest Abroad http://seekingalpha.com/a/1cw2v

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Complaint Review: Meaningful Beauty
Meaningful Beautywww.meaningfulbeauty.com Nationwide U.S.A.
Meaningful Beauty Deceptive practice when I requested cancellation of order Internet
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I called 2 more times on succeeding days and was told the same thing, that my order was not in the system yet and so it could not be cancelled. I was assured that my order would not be shipped yet.
The day after, I saw an email notification that my order had been shipped! I called to protest and was told that they ship the order BEFORE putting it in the computer. I was told that if I returned the shipment I would be reimbursed but it would take 4-6 weeks.
I received the shipment and refused it. Now I am waiting for my refund. I protest that I was denied my right to cancel because the customer service rep I spoke to did not inform me that my order would be shipped BEFORE it was in the system.
Janet xxxxx
Los Angeles, California
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Finance Glossary
Just call us Bond. Amortized bond.
Over 700 finance terms, Shmooped to perfection.
Index Options
Options on an index fund.
You pay for these options so that you can buy or sell an index fund for a specific price by a specific date. You can use index options, just like stock options, as another way to invest money and make money on index funds.


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Reasons You Should Volunteer as a Big Sister
If you're trying to decide if you should volunteer as a Big Sister or Big Brother, here are a few thoughts that might help you decide if it's worth your time.
My Little Sister and I went to the Big Brothers picnic in Vancouver today. My husband is a Big Brother, I worked with Big Brothers/Big Sisters organizations in Vancouver and Calgary, and I was a Big Little Sister when I was 11 years old. I still see my Big Sister, after 35 years! Amazing.
I've never been big on the whole "this is my life" aspect of blogging – I'm definitely a "quips and tips" gal! I write articles to help people achieve goals, solve problems, and move forward in their lives.
But I want to share a few reasons to volunteer as a Big Sister or Big Brother. My Little Sister had a huge impact on my life – I feel better about who I am and what I'm doing in this world. If you're already a Big Sister, read Gift Ideas for Your Sister – Nifty and Thrifty 🙂
What I Learned On My First Outing With My Little Sister
I'm a professional blogger (ie, I make money from my blogs), and my first outing with my new Little Sister has inspired me to let my personal life creep into my blogs.
Here are a few reasons you should volunteer as a Big Sister or Big Brother, based on my experience with my Little Sister at both the Big Brothers and the Big Sisters picnic. The impact you have on a child's life is amazing.
Little Sisters watch everything you say and do
I asked for extra watermelon at the Big Sisters picnic, and so did she. I chose a particular type of dessert, and so did she. I marched around asking Big and Little Sisters questions about the "Get to Know You" bingo game, and so did she. Mentoring – which is what Big Sisters and Big Brothers is all about – means you're constantly being watched.
Is this a reason to volunteer as a Big Sister or Big Brother? Yes, because you learn what a huge impact you have on someone's life, and how important role modeling is.
Volunteer Big Sisters need to walk the walk – not talk the talk
I can lecture my Little Sister about manners, life skills, and social interaction until I'm blue in the face, and not have any affect on her. But, if I make a point of saying thank you to bus drivers, flowing with schedule changes, not getting miserable when it rains during the Big Sisters picnic, and not criticizing her family — that's where I'll make a difference in her life.
Having fun together is life-changing
All I remember about being a Little Sister was roller skating (it was the 80's, after all), going to a scary movie at the drive in, and going camping. A solid, life-changing mentoring relationship doesn't have to be about serious heart-to-heart talks about careers, finances, or life decisions. Changing your Little Sister's life is about showing up once a week, and being enthusiastic about whatever you're doing. Even if you're eating cold hot dogs in the pouring rain.
Big Sisters get creative
When you're first starting a match with Big Sisters or Big Brothers, you should immediately start your scrapbook. My Little Sister and I are talking about what our scrapbook will look like, what we'll include, and how big it should be. We met a match at the Big Sisters picnic who have just celebrated their first year together, but couldn't remember most of the activities they did. That's why a scrapbook is crucial! They've decided to start a Second Year Scrapbook, and regret not starting one when they were first matched.
Learning how to be a kid again is awesomeness!!!
We watched a magic show at the Big Sisters picnic that we went to as our first outing, and on the way back I thought my Little Sister say it wasn't that good. I said agreed with her – I could sorta see how the magician was doing his tricks. But my Little Sister said she thought he WAS a good magician! And here I was, cutting down the magician. I felt horrible, like I'd stolen a big of the magic away. So, on our first outing as Big and Little Sister, I realized that I need to embrace the magic, enthusiasm, and wonder of a child.
Being a Big Sister changes how you see yourself
Research shows mentoring increases the Little Sister or Little Brother's self-confidence and self-esteem. But, does research reveal how it impacts the Big Sisters and Big Brothers? I don't know. I do know that one of my Big Brothers (I'm currently working as a Mentoring Coordinator with Big Brothers of Greater Vancouver, which I wrote about in my post about being scared to quit my job) says that being a Big has changed his life.
Are you thinking about volunteering as a Big Sister or Big Brother because you can't have kids? Read 5 Ideas for Women Who Are Coping With Childlessness.
If you have any questions or thoughts about voluneering as a Big Sister or Big Brother, please comment below!
Related to Your Search
Thank you, Big Sisters of BC Lower Mainland, for the picnic, for matching me with my Little Sister, and for all the good you do in girls' and womens' lives.


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Greater Distance—Greater Flexibility
When used as a stand-alone application extender kit, the B130-111A extends a 1920 x 1440 (60Hz) signal up to 500 ft., or a 1024 x 768 (60Hz) signal up to 1,000 ft, all with premium stereo audio. By inserting a remote/repeater unit between the B130-111A's units, the signal can be regenerated and extended to another set of monitor and speakers up to an additional 1,000 ft. away. Up to three remote/repeater units, like Tripp Lite's B132-110A, can be added to the application, as well as a remote receiver unit, like Tripp Lite's B132-100A, in order to create a chain of up to 2,000 ft. with five monitors and sets of speakers.
Tripp Lite Provides the Complete Solution
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The B130-111A includes mounting hardware, enabling it to be installed in any venue. Mounting options include wall-mount, rackmount and pole mount. For further flexibility, up to three units can be mounted in 1U of rack space using Tripp Lite's B132-004-RB 19-inch rackmount bracket.
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6,169 words (≈ 25 minutes)
Legion of Super Heroes: Cataclysm.
FINAL REGULAR ISSUE! The Conclusion of the Dark Circle Saga is here! A random act of horrible violence sends the Legion of Super-Heroes on a collision course with the Dark Circle. Will the Legion stop the Dark Circle before it's too late? The world shattering conclusion of this series sets the stage for the future of the Legion of Super-Heroes!
and if it weren't bad enough: THIS FINAL BATTLE WILL CLAIM THE LIFE OF AT LEAST ONE FAN-FAVORITE LEGIONNAIRE!
Language: English
Written in: 2007
Published: 2010-08-19
Word count: 6,169 words (≈ 25 minutes)
Tags: Emerald Dragon, Phantom Girl, Cosmic Boy, Lightning Lad, Nemesis Kid, Saturn Girl, Saturn Girl, Brainiac V, Brainiac 5, Polar Boy, Triplicate Girl, Sun Boy, Comics, DC2, Vidar, Blok, Dawnstar
This list contains all the DC2 and DC3 appearances of the Legion of Super Heroes.


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|home | daily thought | life stories | poems | death | loss | testimonials | products
Motivational and Inspirational Stories
It just sorta happens doesn't it... even when there's nothing special about the incident or day? Often times, more cumulative effect, it's not attributable to a single cause. Whatever the drivers, the end result is the same. You find yourself in the early stages of feeling out of sync and alone, feeling that life's prospects don't offer a lot of hope, sensing that none of life's ventures seem to be headed for success.
Perhaps you are even farther along. You can't understand what's wrong with you - and why you can't shake these feelings. Perhaps you have even begun to convince yourself that this is all just a necessary part of life.
Well, the good news is it doesn't have to be. As it turns out, most times the way we feel about situations, people and things is all about our perspectives - and the resulting attitudes and responses applied. As a result, we simply can't expect to have good results if our perspective is in short supply.
Think of it in terms of a silo. Regardless of the amount of grain we start out with, if circumstances cause us to continuously deplete the surplus, with no accompanying replenishment of supply - it's just a matter of time before the silo will have the ability to feed no more. Well, by the same token, if the replenishment of perspective never takes place when times are relatively good, how is it we can expect to have an ample supply to see us through the hard times that will inevitably surface? And for that matter, how is it we could expect the good times to be optimally rewarding.
So, how do we replenish our supply? This is best accomplished by regularly exposing one's psyche to constructive thoughts and anecdotes - which have the power to elevate, enlighten, encourage, impassion, reinforce and reinvigorate both our attitude and spirit. Which is exactly where we come in. Each successive visit and consideration of the perspective featured within the pages of SomethingToShare.com should serve to further replenish your reserves and, accordingly, better equip you to effectively address the bad times - and appreciate the good ones.
So... looking to see things in a better light and to share in a little perspective? Great. Whether this is your first visit or you're a long standing member of the SomethingToShare family... Be inspired. You've come to the right place...
And before you leave, consider taking a little time to tell us what you think, or even better, share your own inspirational story. It will likely prove to be not only therapeutic for you, but also immensely helpful to someone else.
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One Step Back, Two Steps Forward ... (a better tomorrow)
Has a negative circumstance ever entered your otherwise happy and content world? Whether deed or happenstance, has that negativity ever left you feeling drained... frustrated... asking "why me"? And when you felt that way, did those feelings go unresolved? < More >
Kiana ... (better than perfect)
I was six months pregnant and fell off a step stool. It was only two small steps from the floor, so I thought No Big Deal. When I got to the hospital, I was four centimeters dilated and was admitted for observation for two days. On the second day, my water broke and I was put on bed rest for the remainder of my pregnancy in the High Risk Unit. < More >
Chasing Rainbows ... (a new outlook)
A few years back, after a long work week and a rain drenching storm, I found myself stuck in heavy traffic going no where fast. Although there was a chance I would be late for an appointment, I realized that getting irritated wouldn't serve a purpose - or get me there sooner. It was in that frame of mind that I saw it. < More >
What Have I Learned ... (analyzing a life)
The question has been posed with the expectation of an answer - What have you learned from life? This question has been asked by a good friend and requires a thoughtful response. As I ponder the gravity of this question, I realize that a life is the summation of the people we encounter and our experiences involving those people. In recognizing this, I have formulated my answer. < More >
The Look ... (finding the passion again)
Since I can remember, I've always been the kind of person who knew what I wanted in life. My personality was upbeat my outlook and aspirations were eclipsed only by confidence in my abilities and myself. Life held unlimited surprises. The future was an open canvas just waiting for me to supply form, vibrancy and passion. Then my mother passed away. < More >
The Best Thing ... (how we respond)
Newspapers from my hometown said there had been a rise in suicides. Crisis centers were straining. Thousands were suddenly out of work. Factories that had given Akron its nickname, "The Rubber Capitol of the World," were shutting down. No retirement would go to those who dreamed mainly of it. < More >
Jump Out Of Your Boat ... (helping others)
Five fluke in the box, the water was clear, and the sun was shining. It was a hot July day a couple of years ago at Barnegat Inlet, on the coast of New Jersey. This was one of those rare days when the water of the Atlantic Ocean was filling the bay with an incoming tide of crystal clear, almost Caribbean blue beauty. Life was good. < More >
Back To Normal ... (another perspective on 9-11)
We have to get back to normal. That was the popular sentiment. From the President to countless dignitaries, self proclaimed grief and terrorism 'experts' and so many television reporters - everyone concurred the most effective way to respond to the horrific events of September 11, 2001 was to simply get back to normal. < More >
The Well-Dressed Pastor's Lesson ... (remembering roots)
The pastor of the church I attended as a young man was a distinguished, dignified and always impeccably dressed man who also happened to have a warm and compassionate heart. He was so formal and well-groomed that newcomers would expect this tall, handsome man with a PhD from an Ivy League school serving a large, affluent suburban church to be cold and distant. But he wasnt; he was warm and sincere. < More >
Losing My Mother ... (realizing too late)
When I was little, I remember always trying to please my mother. Not that I was an angel, but as a little girl I wanted so much to be the apple of her eye. < More >
Dodgeball Anyone? ... (a weakened society)
They're trying to ban Dodge-ball in schools. Yes Dodge-ball. That simple little game we played as children long ago. While there are variations to the game, the object remains to avoid being hit by the ball. Apparently the people trying to eliminate it's existence from schools and playgrounds across the country believe the game to be too harsh. < More >
Winners And Losers ... (why we sometimes lose)
Why do we concentrate so much on the negative, while spending little time on the positive? Why don't we celebrate more and commiserate less? Why are ninety-five percent of lead news stories covering some combination of ill will, disaster and misfortune? Why does misery love company? < More >
Being A Color ... (getting past the color lines)
It started out as a casual conversation. My friend had mentioned in a previous discussion that she was irritated by an interaction with her daughter's teacher's assistant at the elementary school. Her daughter was reprimanded for hitting another student - explaining to no avail she was only retaliating for being hit. < More >
Thoughts About A Tragedy ... (we often reap what we sow)
Much of this was written the week after the shooting at Columbine High School. While it was a very tragic incident, it inspired me to take a different outlook on life. < More >
A Bump On The Head ... (an amazing fight for life)
Be aware that what I am going to say is 100% fact and is very gruesome. I need to tell you all the gory details so you can truly understand what a miracle it is that I am alive today. On Sunday 11/3/96, I bumped my head on the doorframe while exiting from my car. It stung a bit for a few moments and I thought nothing else about it. < More >
Life Impressions
The More Things Change ... (finally getting it)
I'm sitting here, looking out the window, on what has become a quiet, hot Saturday afternoon. The rain falls, the trees sway, the thunder roars and an occasional streak of lighting makes its way across an otherwise somewhat dreary sky. As my Momma used to tell us this is the Lord's time. < More >
Heavenly Gain ... (appreciating a lost friend)
April 1996 was the start of many things to change my life. The twenty-sixth of April I received a page from my cousin to inform me that my best friend passed away just the night before. At that very moment my life was turned upside down. < More >
In Time ... (a little perspective for a new generation)
When was the last time you felt powerless? Have you ever really felt lost? Ignored? Victimized? Forgotten? When was the last time you felt alone? Imagine how overwhelming it would be to feel that way on a regular basis. Imagine the despair the helplessness. Hard to comprehend being there, huh? Maybe we will in time. < More >
Changed ... (what's wrong with today's youth)
Let me start out by saying I do not have children of my own. On some levels I guess that makes me just about as qualified as many of the recognized modern day experts on the subject. To be perfectly honest, this is an article I felt I wasn't qualified to write - until a good friend, a mother of two, suggested I do so. < More >
Barbara's Story ... (a soul mate story)
On my short ride to work one morning I turned on the radio just in time to hear a listener call in to weigh in on that morning's topic - the existence of soul mates. The woman committed that she knew without a doubt from personal experience that soul mates existed - because she had found and lost hers. < More >
No Laughing Matter ... (a matter of trust)
My girlfriend doesn't like to be tickled. After a long hiatus, her feelings about it resurfaced once again recently as a major point of contention. While playing around, as we do from time to time, I made the apparently grave mistake of tickling her. In response to the cold reaction received from her, my mood took a one hundred and eighty degree turn. < More >
Meantime Blues ... (waiting for life to happen)
As I sit here this evening on a rare moment that I've allowed myself to forget about work, I know I should be sleeping because I have such a busy day planned for tomorrow, but I'm filled with a realization, as well as some questions about my life. < More >
The Event ... (death changes a life)
Recently I watched a movie in which an interesting statement was made. The gist was that there are events that change your life. Nothing is the same afterward. From that point in time every other event is perceived as occurring chronologically before or after the life altering one. I listened quietly and nodded. < More >
Looking Up ... (finding hope again)
Four years ago I was on top of the world. I was a young man - a college graduate, in good health, in a rewarding and well paying career, with a positive attitude and confidence that didn't waiver. < More >
Any Day Now ... (coming to terms with death)
Almost all of my life I've had a fear of death. Thinking back on it today I believe more than anything it was the fear of the unknown. It was troubling to me that the destination was uncertain. < More >
Dealing With Death/Loss
In The Upper Room ... (Mom succumbs)
A close friend has an aunt who, after months of proving a worthy adversary, appears to be succumbing to an increasingly debilitating bout with Cancer. Unfortunately, many of the familiar signs are there - the inhumanity of late night Emergency Room visits extended periods without the consumption of food < More >
The Terrible Incident ... (too deep, the pain)
I always heard tragic stories on the news, in magazines etc., but I never thought anything like that would happen to me. Now I realize that it can happen to anyone even if it is rare. < More >
It Was Just Time ... (too young to go)
I hope my story can help others who have lost someone who has passed at an early age... < More >
Comfy ... (a comforting, lasting link)
Every morning, before I face the world, I kneel next to where you lay. And with one hand on the Bible and the other on you, I pray. I pray for the world for my Mom for me. In you I find comfort. With you I find the strength to face another day. < More >
A Higher Purpose ... (a mother's sacrifice)
This article is to honor my first two children, Charron Latonya and Tinisha Marie and their contribution to this world. < More >
Too Late ... (letting loved ones know)
Before January 12th 1997, I never knew what it was to feel completely empty inside. I'm a 24 year old woman, married, with two kids. I have a good job and pretty happy with my life. So what am I complaining about huh? Let me explain. < More >
You can choose the following link for randomly selected inspirational stories - and see what perspective fate has in store for you today;
go to Inspirational Stories Passage
You can choose the following link to experience a special sampling of some of the wonderfully inspired stories and passages featured in D Anthony's motivational and inspirational book, "The Nurse in the Delivery Room Slapped Me... Once: Stories and Perspectives to Help You Unlock Your Amazing Potential" - a number of which were originally featured here at SomethingToShare.com;
go to featured D Anthony Inspirational Book Excerpts
Or you can choose one of the following links to select inspirational stories from one of our specialty pages;
Dealing with Death, Loss and Grief Page
Faith and Spiritual Foundation Page
Inspiration and Motivation Page
Be Sure To Click Like To Get Updates On Facebook!
Your thoughts on 'Motivational and Inspirational Stories'
Earlier this year I lost a good friend who committed suicide. I later became extremely depressed. My sister passed of cancer. None of my family knew how ill she was. Then to top it off two weeks after her death, I had a head on collision (thank God no one lost their life or was seriously hurt). All this took place in a three month period.
Yes this is a lot to go through. I, I made it through with family, friends and doctor's support. However with all the support and love I had, it was up to me to decide I could make it. And in this journey of life we all go through, some more than others, you must be strong and hold on. There is a light at the end of the tunnel.
My good days - they really out-weigh my bad days. I have learned the real meaning of true friendship and love.
We only have minutes. Take every waking moment you have and appreciate life! Be kind to someone. Love yourself. On this planet, make family first. You may not have the family you want, however you have the family the almighty decided you should have. Love them respect them. Give them their roses while they live. For the next minute is not promised to any of us. Encourage yourself and others too - for God will take care of you.
© 1999 - D-Rose Impressions LLC, All Rights Reserved
and the world will be a better place... one inspired soul at a time


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equipped to crawl since its chassis is shorter than a Slash?
minitrux.net 4x4VXL Slash Rustler & VCreeper
You could tweak and modify it to crawl better than it does out of the box but it will never crawl on the same level as a dedicated purpose built crawler does.
BlindMan Racing
Rusty - MERV - Pede 4x4-Alias
SPC Lipo Power
By golly i think you might be on to something. Brushed motor (or brushless crawler). Locked diffs. Solid axles would be nice but it should be ok. First things first. New rally car. 1/10th scale.Originally Posted by 3traxx


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Opened 7 years ago
Closed 7 years ago
Last modified 18 months ago
#11365 closed Uncategorized (fixed)
django.db.models.fields.EmailField max_length should be 254
|Reported by:
|Owned by:
|Database layer (models, ORM)
|email field max_length
|Triage Stage:
|Has patch:
|Needs documentation:
|Needs tests:
|Patch needs improvement:
|Easy pickings:
http://www.eph.co.uk/resources/email-address-length-faq/ points out that the current max_length of 75 on EmailField fields is too short. It should be extended to 254 to comply with standards.
I checked the EmailField form field, and it does not seem to enforce a min_length or max_length by default, even though the min_length is 6 chars, and max_length is 254.
Attachments (1)
Change History (8)
Changed 7 years ago by shellsage
Patch for correct EmailField max_length, against r11092
comment:1 Changed 7 years ago by graham_king
- Needs documentation unset
- Needs tests unset
- Patch needs improvement unset
+1 because 75 chars is too short to store the Facebook proxied email address that their API dishes out.
comment:2 Changed 7 years ago by russellm
- Resolution set to wontfix
- Status changed from new to closed
Firstly, EmailField accepts a max_length argument, so you can set your email fields to be as long as you like.
Secondly, while I agree that 75 characters isn't strictly standards compliant, it isn't something we can change easily. Changing max_length would be backwards incompatible for every existing installation of Django.
So - when we start having v2.0 discussions, this is one of the backwards incompatible changes will can consider. In the interim, use the max_length argument.
I'm closing this ticket because we don't really have a good way to track 'things we need to do for v2.0'.
comment:3 Changed 7 years ago by anonymous
- Resolution wontfix deleted
- Status changed from closed to reopened
The issue with this is that the EmailField is used by the django auth system, so that is directly affected.
comment:4 Changed 7 years ago by dc
- Resolution set to wontfix
- Status changed from reopened to closed
Please don't reopen tickets that have been closed by a core committer. If you disagree with a decision, start a thread on django-developers.
comment:5 Changed 7 years ago by anonymous
- Cc bas@… added
comment:6 Changed 18 months ago by hheimbuerger
- Easy pickings unset
- Severity set to Normal
- Type set to Uncategorized
- UI/UX unset
This has actually been implemented on the 1.8 branch: https://github.com/django/django/commit/7fd55c3481a004afb049e15ae3b8c93ce8bf0603
Can we mark this ticket as resolved now? It's confusing to see it as marked 'wontfix' when the title actually reflects the current state of the source.
Note: I didn't touch or mean to affect any of the flags Trac just modified…
comment:7 Changed 18 months ago by claudep
- Resolution changed from wontfix to fixed


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I have four questions.
1. This theme: http://wp-themes.com/bible-scholar/
Has three columns. I want to make it 2 columns. One side bar on right side.
- how to do that
2. this theme doesn't have adsense. http://wp-themes.com/simplish/
How to make it adsense ready.
3. If I want to post without date, what code changes do I need to make
4. what is the best source for learning how to customize the wordpress theme.


text:
- Report: #411497
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Complaint Review: RAMADA PLAZA RESORTS
RAMADA PLAZA RESORTS2419 E. COMERCIAL BLVD, SUITE 100 FT. LAUDERDALE, Florida U.S.A.
RAMADA PLAZA RESORTS RIPPOFF FT. LAUDERDALE Florida
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This report was posted on Ripoff Report on 01/13/2009 10:35 AM and is a permanent record located here: http://www.ripoffreport.com/r/RAMADA-PLAZA-RESORTS/FT-LAUDERDALE-Florida-33308/RAMADA-PLAZA-RESORTS-RIPPOFF-FT-LAUDERDALE-Florida-411497. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
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Motorhead, Anthrax, Slayer and More Rock 2015 Motorboat Cruise: Photo Gallery
Here's a gallery of images from Motorhead's Motorboat Cruise, which also featured Anthrax, Slayer, Corrosion of Conformity and others.
Here's a gallery of images from Motorhead's Motorboat Cruise, which also featured Anthrax, Slayer, Corrosion of Conformity and others.
Tina Araya, the mother of Slayer lead singer Tom Araya, has died.
A bizarre murder trial currently unfolding in Maine took an even stranger turn yesterday, when the defendant stood before a judge and claimed that the members of Slayer were somehow involved in the killing.
Slayer rescued a kitten before their concert in Indianapolis on Thursday (Dec. 4).
Sure, you love your favorite bands. But how far would you be willing to go in order to see them in concert?
Rob Scallon performed covered Slayer's 'Raining Blood' on a banjo.
Any tribute that tries to be as comprehensive as the Grammy Awards' annual 'In Memoriam' segment is bound to leave a few people out. But when the Recording Academy aired its remembrance of the musicians we lost in 2013, rock fans noticed one particularly glaring omission: Slayer guitarist Jeff Hanneman.
We jump outside our usual classic rock realm to bring you the most glorious Christmas light show we've seen all year. This eight-minute-plus tribute to Slayer guitarist Jeff Hanneman, who passed away earlier this year, is a must for any hard rock fan.