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Transition metals like Zn are essential for all organisms including bacteria, but fluctuations of their concentrations in the cell can be lethal. Organisms have thus evolved complex mechanisms for cellular metal homeostasis. One mechanistic paradigm involves pairs of transcription regulators sensing intracellular metal concentrations to regulate metal uptake and efflux. Here we report that Zur and ZntR, a prototypical pair of regulators for Zn uptake and efflux in E. coli , respectively, can coordinate their regulation through DNA, besides sensing cellular Zn 2+ concentrations. Using a combination of live-cell single-molecule tracking and in vitro single-molecule FRET measurements, we show that unmetallated ZntR can enhance the unbinding kinetics of Zur from DNA by directly acting on Zur-DNA complexes, possibly through forming heteromeric ternary and quaternary complexes that involve both protein-DNA and protein-protein interactions. This 'through-DNA' mechanism may functionally facilitate the switching in Zn uptake regulation when bacteria encounter changing Zn environments; it could also be relevant for regulating the uptake-vs.-efflux of various metals across different bacterial species and yeast.
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PURPOSE: Electromagnetic (EM) catheter tracking has recently been introduced in order to enable prompt and uncomplicated reconstruction of catheter paths in various clinical interventions. However, EM tracking is prone to measurement errors which can compromise the outcome of the procedure. Minimizing catheter tracking errors is therefore paramount to improve the path reconstruction accuracy. METHODS: An extended Kalman filter (EKF) was employed to combine the nonlinear kinematic model of an EM sensor inside the catheter, with both its position and orientation measurements. The formulation of the kinematic model was based on the nonholonomic motion constraints of the EM sensor inside the catheter. Experimental verification was carried out in a clinical HDR suite. Ten catheters were inserted with mean curvatures varying from 0 to [Formula: see text] in a phantom. A miniaturized Ascension (Burlington, Vermont, USA) trakSTAR EM sensor (model 55) was threaded within each catheter at various speeds ranging from 7.4 to [Formula: see text]. The nonholonomic EKF was applied on the tracking data in order to statistically improve the EM tracking accuracy. A sample reconstruction error was defined at each point as the Euclidean distance between the estimated EM measurement and its corresponding ground truth. A path reconstruction accuracy was defined as the root mean square of the sample reconstruction errors, while the path reconstruction precision was defined as the standard deviation of these sample reconstruction errors. The impacts of sensor velocity and path curvature on the nonholonomic EKF method were determined. Finally, the nonholonomic EKF catheter path reconstructions were compared with the reconstructions provided by the manufacturer's filters under default settings, namely the AC wide notch and the DC adaptive filter. RESULTS: With a path reconstruction accuracy of 1.9 mm, the nonholonomic EKF surpassed the performance of the manufacturer's filters (2.4 mm) by 21% and the raw EM measurements (3.5 mm) by 46%. Similarly, with a path reconstruction precision of 0.8 mm, the nonholonomic EKF surpassed the performance of the manufacturer's filters (1.0 mm) by 20% and the raw EM measurements (1.7 mm) by 53%. Path reconstruction accuracies did not follow an apparent trend when varying the path curvature and sensor velocity; instead, reconstruction accuracies were predominantly impacted by the position of the EM field transmitter ([Formula: see text]). CONCLUSION: The advanced nonholonomic EKF is effective in reducing EM measurement errors when reconstructing catheter paths, is robust to path curvature and sensor speed, and runs in real time. Our approach is promising for a plurality of clinical procedures requiring catheter reconstructions, such as cardiovascular interventions, pulmonary applications (Bender et al. in medical image computing and computer-assisted intervention-MICCAI 99. Springer, Berlin, pp 981-989, 1999), and brachytherapy.
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Braquiterapia/métodos , Catéteres , Programas Informáticos , Fenómenos Electromagnéticos , Humanos , Fantasmas de ImagenRESUMEN
FtsZ, a bacterial tubulin homologue, is a cytoskeletal protein that assembles into protofilaments that are one subunit thick. These protofilaments assemble further to form a "Z ring" at the center of prokaryotic cells. The Z ring generates a constriction force on the inner membrane and also serves as a scaffold to recruit cell wall remodeling proteins for complete cell division in vivo One model of the Z ring proposes that protofilaments associate via lateral bonds to form ribbons; however, lateral bonds are still only hypothetical. To explore potential lateral bonding sites, we probed the surface of Escherichia coli FtsZ by inserting either small peptides or whole fluorescent proteins (FPs). Among the four lateral surfaces on FtsZ protofilaments, we obtained inserts on the front and back surfaces that were functional for cell division. We concluded that these faces are not sites of essential interactions. Inserts at two sites, G124 and R174, located on the left and right surfaces, completely blocked function, and these sites were identified as possible sites for essential lateral interactions. However, the insert at R174 did not interfere with association of protofilaments into sheets and bundles in vitro Another goal was to find a location within FtsZ that supported insertion of FP reporter proteins while allowing the FtsZ-FPs to function as the sole source of FtsZ. We discovered one internal site, G55-Q56, where several different FPs could be inserted without impairing function. These FtsZ-FPs may provide advances for imaging Z-ring structure by superresolution techniques. IMPORTANCE: One model for the Z-ring structure proposes that protofilaments are assembled into ribbons by lateral bonds between FtsZ subunits. Our study excluded the involvement of the front and back faces of the protofilament in essential interactions in vivo but pointed to two potential lateral bond sites, on the right and left sides. We also identified an FtsZ loop where various fluorescent proteins could be inserted without blocking function; these FtsZ-FPs functioned as the sole source of FtsZ. This advance provides improved tools for all fluorescence imaging of the Z ring and may be especially important for superresolution imaging.
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Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Mutagénesis Sitio-Dirigida , Proteínas Bacterianas/genética , Proteínas del Citoesqueleto/genética , Escherichia coli/genética , Fluorescencia , Modelos Moleculares , Mutagénesis Insercional , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
Metalloregulators respond to metal ions to regulate transcription of metal homeostasis genes. MerR-family metalloregulators act on σ(70)-dependent suboptimal promoters and operate via a unique DNA distortion mechanism in which both the apo and holo forms of the regulators bind tightly to their operator sequence, distorting DNA structure and leading to transcription repression or activation, respectively. It remains unclear how these metalloregulator-DNA interactions are coupled dynamically to RNA polymerase (RNAP) interactions with DNA for transcription regulation. Using single-molecule FRET, we study how the copper efflux regulator (CueR)--a Cu(+)-responsive MerR-family metalloregulator--modulates RNAP interactions with CueR's cognate suboptimal promoter PcopA, and how RNAP affects CueR-PcopA interactions. We find that RNAP can form two noninterconverting complexes at PcopA in the absence of nucleotides: a dead-end complex and an open complex, constituting a branched interaction pathway that is distinct from the linear pathway prevalent for transcription initiation at optimal promoters. Capitalizing on this branched pathway, CueR operates via a "biased sampling" instead of "dynamic equilibrium shifting" mechanism in regulating transcription initiation; it modulates RNAP's binding-unbinding kinetics, without allowing interconversions between the dead-end and open complexes. Instead, the apo-repressor form reinforces the dominance of the dead-end complex to repress transcription, and the holo-activator form shifts the interactions toward the open complex to activate transcription. RNAP, in turn, locks CueR binding at PcopA into its specific binding mode, likely helping amplify the differences between apo- and holo-CueR in imposing DNA structural changes. Therefore, RNAP and CueR work synergistically in regulating transcription.
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Adenosina Trifosfatasas/metabolismo , Proteínas de Transporte de Catión/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Algoritmos , Secuencia de Bases , Carbocianinas/química , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , ATPasas Transportadoras de Cobre , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Cinética , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Factor sigma/química , Factor sigma/genética , Factor sigma/metabolismo , Transactivadores/química , Transactivadores/genéticaRESUMEN
Understanding how cells regulate and transport metal ions is an important goal in the field of bioinorganic chemistry, a frontier research area that resides at the interface of chemistry and biology. This Current Topic reviews recent advances from the authors' group in using single-molecule fluorescence imaging techniques to identify the mechanisms of metal homeostatic proteins, including metalloregulators and metallochaperones. It emphasizes the novel mechanistic insights into how dynamic protein-DNA and protein-protein interactions offer efficient pathways via which MerR-family metalloregulators and copper chaperones can fulfill their functions. This work also summarizes other related single-molecule studies of bioinorganic systems and provides an outlook toward single-molecule imaging of metalloprotein functions in living cells.
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Proteínas de Unión al ADN/metabolismo , Metalochaperonas/metabolismo , Metales/metabolismo , Animales , Proteínas de Unión al ADN/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Metalochaperonas/química , Metalochaperonas/genéticaRESUMEN
PURPOSE: Investigations have shown that a Cobalt-60 (Co-60) radioactive source has the potential to play a role in intensity modulated radiation therapy (IMRT). In this paper, Co-60 tomotherapy's conformal dose delivery potential is evaluated by delivering conformal dose plans on a cylindrical homogeneous phantom containing clinical structures similar to those found in a typical head and neck (H&N) cancer. Also, the clinical potential of Co-60 tomotherapy is investigated by generating 2D clinical treatment plans for H&N and prostate anatomical regions. These plans are compared with the 6 MV based treatment plans for modalities such as linear accelerator-based tomotherapy and broad beam IMRT, and 15 MV based 3D conformal radiation therapy (3DCRT). METHODS: For experimental validation studies, clinical and nonclinical conformal dose patterns were delivered on circular, homogeneous phantoms containing GafChromic film. For clinical planning study, dose calculations were performed with the EGSnrc Monte Carlo program, where a Theratronics 780C Co-60 unit and a 6 MV linear accelerator were modeled with a MIMiC binary multileaf collimator. An inhouse inverse treatment planning system was used to optimize tomotherapy plans using the same optimization parameters for both Co-60 and 6 MV beams. The IMRT and 3DCRT plans for the clinical cases were generated entirely in the Eclipse treatment planning system based on inhouse IMRT and 3DCRT site specific protocols. RESULTS: The doses delivered to the homogeneous phantoms agreed with the calculations, indicating that it is possible to deliver highly conformal doses with the Co-60 unit. The dose distributions for Co-60 tomotherapy clinical plans for both clinical cases were similar to those obtained with 6 MV based tomotherapy and IMRT, and much more conformal compared to 3DCRT plans. The dose area histograms showed that the Co-60 plans achieve the dose objectives for the targets and organs at risk. CONCLUSIONS: These results confirm that Co-60 tomotherapy is capable of providing state-of-the-art conformal dose delivery and could be used for the treatment of targets in both small and larger separation anatomical regions.
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Fantasmas de Imagen , Dosis de Radiación , Planificación de la Radioterapia Asistida por Computador/instrumentación , Radioterapia Asistida por Computador/métodos , Radioisótopos de Cobalto/uso terapéutico , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Masculino , Método de Montecarlo , Neoplasias de la Próstata/radioterapia , Dosificación RadioterapéuticaRESUMEN
Metalloregulators regulate transcription in response to metal ions. Many studies have provided insights into how transcription is activated upon metal binding by MerR-family metalloregulators. In contrast, how transcription is turned off after activation is unclear. Turning off transcription promptly is important, however, as the cells would not want to continue expressing metal resistance genes and thus waste energy after metal stress is relieved. Using single-molecule FRET measurements we studied the dynamic interactions of the copper efflux regulator (CueR), a Cu(+)-responsive MerR-family metalloregulator, with DNA. Besides quantifying its DNA binding and unbinding kinetics, we discovered that CueR spontaneously flips its binding orientation at the recognition site. CueR also has two different binding modes, corresponding to interactions with specific and nonspecific DNA sequences, which would facilitate recognition localization. Most strikingly, a CueR molecule coming from solution can directly substitute for a DNA-bound CueR or assist the dissociation of the incumbent CueR, both of which are unique examples for any DNA-binding protein. The kinetics of the direct protein substitution and assisted dissociation reactions indicate that these two unique processes can provide efficient pathways to replace a DNA-bound holo-CueR with apo-CueR, thus turning off transcription promptly and facilely.
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Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Iones/química , Fenómenos Fisiológicos Bacterianos , ADN/química , Proteínas de Unión al ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Metales/química , Modelos Biológicos , Unión Proteica , Compuestos de Sulfhidrilo/química , Transcripción GenéticaRESUMEN
Underdosing of treatment targets can occur in radiation therapy due to electronic disequilibrium around air-tissue interfaces when tumors are situated near natural air cavities. These effects have been shown to increase with the beam energy and decrease with the field size. Intensity modulated radiation therapy (IMRT) and tomotherapy techniques employ combinations of multiple small radiation beamlets of varying intensities to deliver highly conformal radiation therapy. The use of small beamlets in these techniques may therefore result in underdosing of treatment target in the air-tissue interfaces region surrounding an air cavity. This work was undertaken to investigate dose reductions near the air-water interfaces of 1x1x1 and 3x3x3 cm(3) air cavities, typically encountered in the treatment of head and neck cancer utilizing radiation therapy techniques such as IMRT and tomotherapy using small fields of Co-60, 6 MV and 15 MV photons. Additional investigations were performed for larger photon field sizes encompassing the entire air-cavity, such as encountered in conventional three dimensional conformal radiation therapy (3DCRT) techniques. The EGSnrc/DOSXYZnrc Monte Carlo code was used to calculate the dose reductions (in water) in air-water interface region for single, parallel opposed and four field irradiations with 2x2 cm(2) (beamlet), 10x2 cm(2) (fan beam), 5x5 and 7x7 cm(2) field sizes. The magnitude of dose reduction in water near air-water interface increases with photon energy; decreases with distance from the interface as well as decreases as the number of beams are increased. No dose reductions were observed for large field sizes encompassing the air cavities. The results demonstrate that Co-60 beams may provide significantly smaller interface dose reductions than 6 MV and 15 MV irradiations for small field irradiations such as used in IMRT and tomotherapy.
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In the Y42F mutant of photoactive yellow protein (PYP) the photoreceptor is in an equilibrium between two dark states, the yellow and intermediate spectral forms, absorbing at 457 and 390 nm, respectively. The nature of this equilibrium and the light-induced protonation and structural changes in the two spectral forms were characterized by transient absorption, fluorescence, FTIR, and pH indicator dye experiments. In the yellow form, the oxygen of the deprotonated p-hydroxycinnamoyl chromophore is linked by a strong low-barrier hydrogen bond to the protonated carboxyl group of Glu46 and by a weaker one to Thr50. Using FTIR, we find that the band due to the carbonyl of the protonated side chain of Glu46 is shifted from 1736 cm(-1) in wild type to 1724 cm(-1) in the yellow form of Y42F, implying a stronger hydrogen bond with the deprotonated chromophore in Y42F. The FTIR data suggest moreover that in the intermediate spectral form the chromophore is protonated and Glu46 deprotonated. Flash spectroscopy (50 ns-10 s) shows that the photocycles of the two forms are essentially the same except for a transition around 5 mus that has opposite signs in the two forms and is due to the chemical relaxation between the two dark states. The two cycles are coupled, likely by excited state proton transfer. The Y42F cycle differs from wild type by the occurrence of a new intermediate with protonated chromophore between the usual I(1) and I(2) intermediates which we call I(1)H (370 nm). Transient fluorescence measurements indicate that in I(1)H the chromophore retains the orientation it had in I(1). Transient proton uptake occurs with a time constant of 230 mus and a stoichiometry of 1. No proton uptake was associated however with the formation of the I(1)H intermediate and the relaxation of the yellow/intermediate equilibrium. These protonation changes of the chromophore thus occur intramolecularly. The chromophore-Glu46 hydrogen bond in Y42F is shorter than in wild type, since the adjacent chromophore-Y42 hydrogen bond is replaced by a longer one with Thr50. This facilitates proton transfer from Glu46 to the chromophore in the dark by lowering the barrier, leading to the protonation equilibrium and causing the rapid light-induced proton transfer which couples the cycles.
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Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Ácido Glutámico/química , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Protones , Proteínas Bacterianas/metabolismo , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Enlace de Hidrógeno , Cinética , Mutación , Fotorreceptores Microbianos/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The advances in modern radiation therapy with techniques such as intensity-modulated radiation therapy and image-guided radiation therapy (IMRT and IGRT) have been limited almost exclusively to linear accelerators. Investigations of modern Cobalt-60 (Co-60) radiation delivery in the context of IMRT and IGRT have been very sparse, and have been limited mainly to computer-modeling and treatment-planning exercises. In this paper, we report on the results of experiments using a tomotherapy benchtop apparatus attached to a conventional Co-60 unit. We show that conformal dose delivery is possible and also that Co-60 can be used as the radiation source in megavoltage computed tomography imaging. These results complement our modeling studies of Co-60 tomotherapy and provide a strong motivation for continuing development of modern Cobalt-60 treatment devices.
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Cobalt-60 (Co-60) based radiation therapy continues to play a significant role in not only developing countries, where access to radiation therapy is extremely limited, but also in industrialized countries. Howver, technology has to be developed to accommodate modern techniques, including image guided and adaptive radiation therapy (IGART). In this paper we describe some of the practical and clinical considerations for Co-60 based tomotherapy by comparing Co-60 and 6 MV linac-based tomotherapy plans for a head and neck (HandN) cancer and a prostate cancer case. The tomotherapy IMRT plans were obtained by modeling a MIMiC binary multi-leaf collimator attached to a Theratron-780c Co-60 unit and a 6 MV linear accelerator (CL2100EX). The EGSnrc/BEAMnrc Monte Carlo (MC) code was used for the modeling of the treatment units with the MIMiC collimator and EGSnrc/DOSXYZnrc code was used for beamlet dose data. An in-house inverse treatment planning program was then used to generate optimized tomotherapy dose distributions for the H and N and prostate cases. The dose distributions, cumulative dose area histograms (DAHs) and dose difference maps were used to evaluate and compare Co-60 and 6 MV based tomotherapy plans. A quantitative analysis of the dose distributions and dose-volume histograms shows that both Co-60 and 6 MV plans achieve the plan objectives for the targets (CTV and nodes) and OARs (spinal cord in HandN case, and rectum in prostate case).
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Recent investigations demonstrate a strong potential for Cobalt-60 (Co-60)-based tomotherapy. Reported work suggests that Co-60-based tomotherapy offers a clinically and commercially viable alternative to megavoltage x-ray-based tomotherapy. Tomotherapy applications use a combination of intensity-modulated fan beams to deliver highly conformal radiotherapy. However, conventional Co-60 units are designed to give large uniform rectangular fields using an isotropic radioactive source in a cylindrical geometry. Such cylindrical source geometry likely provides a sub-optimal use of the radioactivity within the source volume for tomotherapy applications due to a significant loss of radiated energy outside the fan beam collimation system. To investigate a more efficient source geometry suitable for Co-60 tomotherapy applications, a computer code was written to model an isotropic source in a 6-faced polyhedron geometry such as cube, parallelepiped, prism and truncated pyramid. This code was integrated with the existing EGSnrc/BEAMnrc Monte Carlo (MC) code. The integrated source code was thoroughly tested, validated and used to investigate the energy spectra, radiation output and self-shielding properties of various rectangular-shaped (RS) Co-60 sources. Fan beam dose profiles were calculated for various cylindrical and RS Co-60 sources for a range of source-to-axis distances (SAD), multi-leaf collimator-to-isocentre distances (CID) and modified collimator systems. Fringe and penumbra distances were analysed for the simulated dose profiles. Our results demonstrate that clinically acceptable fringe and penumbra distances can be achieved by a careful selection of SAD, CID, source shape and dimensions and modified collimator system. Significant overall gain in radiation output of the 20 x 1 cm(2) fan beams can be achieved by an optimal selection of the source geometry for a given active volume of Co-60. The overall gain includes the effects of change in packing density (accounting for self-absorption) and change in source shape.
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Algoritmos , Radioisótopos de Cobalto/uso terapéutico , Diseño Asistido por Computadora , Radiometría/métodos , Planificación de la Radioterapia Asistida por Computador/métodos , Radioterapia Conformacional/instrumentación , Programas Informáticos , Diseño de Equipo , Análisis de Falla de Equipo , Método de Montecarlo , Dosificación Radioterapéutica , Radioterapia Conformacional/métodosRESUMEN
The effect of ionic strength on the conformational equilibrium between the I(2) intermediate and the signaling state I(2)' of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to approximately 600 mM, the recovery rate k(3) decreases and the I(2)/I(2)' equilibrium (K) shifts in the direction of I(2)'. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl(2), MgSO(4)) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k(3) and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be -1.3 +/- 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the beta-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I(2)/I(2)' equilibrium was eliminated and of the recovery rate was greatly reduced below approximately 600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I(2) structure. However, chaotropic anions like SCN shift the I(2)/I(2)' equilibrium toward the partially unfolded I(2)' form. The salt linkage K110/E12 stabilizes the photoreceptor in the inactive state in the dark and is broken in the light-induced formation of the signaling state, allowing the N-terminal domain to detach from the beta-scaffold PAS core.
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Proteínas Bacterianas/química , Biofisica/métodos , Células Fotorreceptoras/metabolismo , Fotorreceptores Microbianos/química , Sales (Química)/farmacología , Aniones , Concentración de Iones de Hidrógeno , Iones , Cinética , Modelos Estadísticos , Mutación , Estructura Terciaria de Proteína , Sales (Química)/química , Transducción de Señal , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
The signaling state of the photoreceptor photoactive yellow protein is the long-lived intermediate I(2)'. The pH dependence of the equilibrium between the transient photocycle intermediates I(2) and I(2)' was investigated. The formation of I(2)' from I(2) is accompanied by a major conformational change. The kinetics and intermediates of the photocycle and of the photoreversal were measured by transient absorption spectroscopy from pH 4.6 to 8.4. Singular value decomposition (SVD) analysis of the data at pH 7 showed the presence of three spectrally distinguishable species: I(1), I(2), and I(2)'. Their spectra were determined using the extrapolated difference method. I(2) and I(2)' have electronic absorption spectra, with maxima at 370 +/- 5 and 350 +/- 5 nm, respectively. Formation of the signaling state is thus associated with a change in the environment of the protonated chromophore. The time courses of the I(1), I(2), and I(2)' intermediates were determined from the wavelength-dependent transient absorbance changes at each pH, assuming that their spectra are pH-independent. After the formation of I(2)' ( approximately 2 ms), these three intermediates are in equilibrium and decay together to the initial dark state. The equilibrium between I(2) and I(2)' is pH dependent with a pK(a) of 6.4 and with I(2)' the main species above this pK(a). Measurements of the pH dependence of the photoreversal kinetics with a second flash of 355 nm at a delay of 20 ms confirm this pK(a) value. I(2) and I(2)' are photoreversed with reversal times of approximately 55 micros and several hundred microseconds, respectively. The corresponding signal amplitudes are pH dependent with a pK(a) of approximately 6.1. Photoreversal from I(2)' dominates above the pK(a). The transient accumulation of I(2)', the active state of photoactive yellow protein, is thus controlled by the proton concentration. The rate constant k(3) for the recovery to the initial dark state also has a pK(a) of approximately 6.3. This equality of the equilibrium and kinetic pK(a) values is not accidental and suggests that k(3) is proportional to [I(2)'].
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Proteínas Bacterianas/fisiología , Halorhodospira halophila/fisiología , Fotorreceptores Microbianos/fisiología , Transducción de Señal/fisiología , Proteínas Bacterianas/química , Halorhodospira halophila/efectos de la radiación , Concentración de Iones de Hidrógeno , Cinética , Luz , Fotorreceptores Microbianos/química , Conformación ProteicaRESUMEN
Since the habitat of Halorhodospira halophila is distinctly alkaline, we investigated the kinetics and intermediates of the photocycle and photoreversal of the photoreceptor photoactive yellow protein (PYP) from pH 8 to 11. SVD analysis of the transient absorption time traces in a broad wavelength range (330-510 nm) shows the presence of three spectrally distinct species (I1, I1', and I2') at pH 10. The spectrum of I1' was obtained in two different ways. The maximal absorption is at 425 nm. I1' probably has a deprotonated chromophore and may be regarded as the alkaline form of I2'. At pH 10, the I1 intermediate decays in approximately 330 micros in part to I1' before I1 and I1' decay further to I2' in approximately 1 ms. From the rise of I2' (approximately 1 ms) to the end of the photocycle, the three intermediates (I1, I1', and I2') remain in equilibrium and decay together to P in approximately 830 ms. Assuming that the spectra of I1, I1', and I2' are pH-independent, their time courses were determined. On the millisecond to second time scale, they are in a pH-dependent equilibrium with a pKa of approximately 9.9. With an increase in pH, the I1 and I1' populations increase at the expense of the amount of I2'. The apparent rate constant for the recovery of P slows with an increase in pH with a pKa of approximately 9.7. The equal pH dependence of this rate and the equilibrium concentrations follows, if we assume that the equilibration rates between the intermediates are much faster than the recovery rate and that the recovery occurs from I2'. The pKa of approximately 9.9 is assigned to the deprotonation of the phenol of the surface-exposed chromophore in the I1'-I2' equilibrium. The I1-I1' equilibrium is pH-independent. Photoreversal experiments at pH 10 with the second flash at 355 nm indicate the presence of only one I2-like intermediate, which we assign on the basis of its lambda(max) value to I2'. After the rapid unresolved photoisomerization to I2'(trans), the reversal pathway back to P involves two sequential steps (60 micros and 3 ms). The amplitude spectra show that I1'(trans) and I1(trans) intermediates participate in this reversal.
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Proteínas Bacterianas/química , Concentración de Iones de Hidrógeno , Fotoquímica , Fotorreceptores Microbianos/química , CinéticaRESUMEN
Automated seed loaders for permanent prostate implants are now commercially available. Besides improved radiation safety, these systems offer seed assay capability and ease of needle loading, making preplanned as well as intra-operative implant procedures more time-efficient. The Isoloader (Mentor Corp., CA) uses individual I125 seeds (SL-125 ProstaSeed) loaded in up to 199 chambers inside a shielded cartridge. The unit performs seed counting and calibration using a builtin solid-state detector. In order to evaluate the reproducibility and accuracy of the calibration process, two test cartridges were measured with the Isoloader itself and compared with a well-type ionization chamber (HDR-1000Plus, Standard Imaging). The air kerma strength measurements for all seeds using the Isoloader had a standard deviation of about 2.7%. For the eight seeds assayed more intensively using both the Isoloader and well chamber, the standard deviations of the measurements for each seed were in the range of 0.8% to 2.8% and 0.6% to 1.3%, respectively. The variation in the Isoloader calibration is attributed to small detector solid angle and bead geometry within seed capsules (verified by radiographs). The reproducibility of the air kerma strength measured by the Isoloader was comparable to that from the well chamber and was clinically acceptable. Seed strength measured with the Isoloader was on average 1% 2% larger than that measured with the well chamber, indicating that the accuracy of the Isoloader was clinically acceptable.
Asunto(s)
Braquiterapia/instrumentación , Agujas , Neoplasias de la Próstata/radioterapia , Implantación de Prótesis/instrumentación , Radiometría/instrumentación , Robótica/instrumentación , Manejo de Especímenes/instrumentación , Braquiterapia/métodos , Calibración , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Masculino , Implantación de Prótesis/normas , Radiometría/métodos , Radiometría/normas , Radioterapia , Robótica/métodos , Manejo de Especímenes/métodos , Manejo de Especímenes/normasRESUMEN
We investigated the photocycle of mutants Y98Q and Y98F of the photoactive yellow protein (PYP) from Halorhodospira halophila. Y98 is located in the beta4-beta5 loop and is thought to interact with R52 in the alpha3-alpha4 loop thereby stabilizing this region. Y98 is conserved in all known PYP species, except in Ppr and Ppd where it is replaced by F. We find that replacement of Y98 by F has no significant effect on the photocycle kinetics. However, major changes were observed with the Y98Q mutant. Our results indicate a requirement for an aromatic ring at position 98, especially for recovery and a normal I1/I2 equilibrium. The ring of Y98 could stabilize the beta4-beta5 loop. Alternatively, the Y98 ring could transiently interact with the isomerized chromophore ring, thereby stabilizing the I2 intermediate in the I1/I2 equilibrium. For Y98Q, the decay of the signaling state I2' was slowed by a factor of approximately 40, and the rise of the I2 and I2' intermediates was slowed by a factor of 2-3. Moreover, the I1 intermediate is in a pH-dependent equilibrium with I2/I2' with the ratio of the I1 and I2 populations close to one at pH 7 and 50 mM KCl. From pH 5.5 to 8, the equilibrium shifts toward I1, with a pKa of approximately 6.3. Above pH 8, the populations of I1 and I2/I2' decrease due to an equilibrium between I1 and an additional species I1' which absorbs at approximately 425 nm (pKa approximately 9.8) and which we believe to be an I2-like form with a surface-exposed deprotonated chromophore. The I1/I2/I2' equilibrium was found to be strongly dependent on the KCl concentration, with salt stabilizing the signaling state I2' up to 600 mM KCl. This salt-induced transition to I2' was analyzed and interpreted as ion binding to a specific site. Moreover, from analysis of the amplitude spectra, we conclude that KCl exerts its major effect on the I2 to I2' transition, i.e., the global conformational change leading to the signaling state I2' and the exposure of a hydrophobic surface patch. In wild type and Y98F, the I1/I2 equilibrium is more on the side of I2/I2' as compared to Y98Q but is also salt-dependent at pH 7. The I2 to I2' transition appears to be controlled by an ionic lock, possibly involving the salt bridge between K110 on the beta-scaffold and E12 on the N-terminal cap. Salt binding would break the salt bridge and weaken the interaction between the two domains, facilitating the release of the N-terminal domain from the beta-scaffold in the formation of I2'.
Asunto(s)
Proteínas Bacterianas/química , Concentración de Iones de Hidrógeno , Mutación , Fotorreceptores Microbianos/química , Sales (Química)/química , Proteínas Bacterianas/genética , Halorhodospira halophila/química , Halorhodospira halophila/genética , Fotoquímica , Fotorreceptores Microbianos/genéticaRESUMEN
We investigated the kinetics of photoreversal from the I(1) and I(2) intermediates of photoactive yellow protein (PYP) by time-resolved optical absorption spectroscopy with double flash excitation. A first flash, at 430 nm, initiated the photocycle. After a variable time delay, the I(1) intermediate was photoreversed by a second flash, at 500 nm, or a mixture of I(2) and I(2)' intermediates was photoreversed by a second flash, at 355 nm. By varying the delay from 1 micros to 3 s, we were able to selectively excite the intermediates I(1), I(2), and I(2)'. The photoreversal kinetics of I(2) and I(2)' at 21 different delays and two wavelengths (340 and 450 nm) required two exponentials for a global fit with time constants of tau(1) = 57 +/- 5 micros and tau(2) = 380 +/- 40 micros (pH 6, 20 degrees C). These were assigned to photoreversal from sequential I(2) and I(2)' intermediates, respectively. The good agreement of the delay dependence of the two amplitudes, A(1) and A(2), with the time dependence of the I(2) and I(2)' populations provided strong evidence for the sequential model. The persistence of A(1) beyond delay times of 5 ms and its decay, together with A(2) around 500 ms, suggest moreover that I(2) and I(2)' are in thermal equilibrium. The wavelength dependence of the photoreversal kinetics was measured at 26 wavelengths from 510 to 330 nm at the two fixed delays of 1 and 10 ms. These data also required two exponentials for a global fit with tau(1) = 59 +/- 5 micros and tau(2) = 400 +/- 40 micros, in good agreement with the delay results. Photoreversal from I(2)' is slower than from I(2), since, in addition to chromophore protonation, the global conformational change has to be reversed. Our data thus provide a first estimate of about 59 micros for deprotonation and 400 micros for the structural change, which also occurs in the thermal decay of the signaling state but is obscured there since reisomerization is rate-limiting. The first step in photoreversal is rapid cis-trans isomerization of the chromophore, which we could not resolve, but which was detected by the instantaneous increase in absorbance between 330 and 380 nm. In agreement with this observation, the spectrum of the I(2)'(trans) intermediate, derived from the A(2) amplitude spectrum, has a much larger extinction coefficient than the spectrum of the I(2)'(cis) intermediate. With a first flash, at 430 nm, and a second flash, at 500 nm, we observed efficient photoreversal of the I(1) intermediate at a delay of 20 micros when most molecules in the cycle are in I(1). We conclude that each of the three intermediates studied can be reversed by a laser flash. Depending on the progression of the photocycle, reversal becomes slower with the time delay, thus mirroring the individual steps of the forward photocycle.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Fotólisis , Fotoperiodo , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Oscuridad , Halorhodospira halophila , Isomerismo , Cinética , Luz , Modelos Químicos , Transducción de Señal , Espectrofotometría , Factores de TiempoRESUMEN
The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.