RESUMEN
Metal-free porphyrin-dendrimers provide a convenient platform for the construction of membrane-impermeable ratiometric probes for pH measurements in compartmentalized biological systems. In all previously reported molecules, electrostatic stabilization (shielding) of the core porphyrin by peripheral negative charges (carboxylates) was required to shift the intrinsically low porphyrin protonation pK(a)'s into the physiological pH range (pH 6-8). However, binding of metal cations (e.g., K(+), Na(+), Ca(2+), Mg(2+)) by the carboxylate groups on the dendrimer could affect the protonation behavior of such probes in biological environments. Here we present a dendritic pH nanoprobe based on a highly non-planar tetraaryltetracyclohexenoporphyrin (Ar(4)TCHP), whose intrinsic protonation pK(a)'s are significantly higher than those of regular tetraarylporphyrins, thereby eliminating the need for electrostatic core shielding. The porphyrin was modified with eight Newkome-type dendrons and PEGylated at the periphery, rendering a neutral water-soluble probe (TCHpH), suitable for measurements in the physiological pH range. The protonation of TCHpH could be followed by absorption (e.g., ε(Soret)(dication)â¼270,000 M(-1) cm(-1)) or by fluorescence. Unlike most tetraarylporphyrins, TCHpH is protonated in two distinct steps (pK(a)'s 7.8 and 6.0). In the region between the pK(a)'s, an intermediate species with a well-defined spectroscopic signature, presumably a TCHpH monocation, could be observed in the mixture. The performance of TCHpH was evaluated by pH gradient measurements in large unilamellar vesicles. The probe was retained inside the vesicles and did not pass through and/or interact with vesicle membranes, proving useful for quantification of proton transport across phospholipid bilayers. To interpret the protonation behavior of TCHpH we developed a model relating structural changes on the porphyrin macrocycle upon protonation to its basicity. The model was validated by density functional theory (DFT) calculations performed on a planar and non-planar porphyrin, making it possible to rationalize higher protonation pK(a)'s of non-planar porphyrins as well as the easier observation of their monocations.
Asunto(s)
Dendrímeros/química , Metaloporfirinas/química , Porfirinas/química , Cationes/síntesis química , Cationes/química , Concentración de Iones de Hidrógeno , Metaloporfirinas/síntesis química , Estructura Molecular , EstereoisomerismoRESUMEN
The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.
Asunto(s)
Virus de la Influenza A/metabolismo , Canales Iónicos/metabolismo , Protones , Proteínas de la Matriz Viral/metabolismo , Algoritmos , Transporte Biológico/efectos de los fármacos , Cationes/metabolismo , Concentración de Iones de Hidrógeno , Virus de la Influenza A/genética , Canales Iónicos/química , Canales Iónicos/genética , Cinética , Modelos Biológicos , Mutación , Potasio/metabolismo , Proteolípidos/química , Proteínas Recombinantes/metabolismo , Sodio/metabolismo , Valinomicina/farmacología , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.
Asunto(s)
Citocromos c/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Citocromos c/química , Citocromos c/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Accurate real-time measurements of proton concentration gradients are pivotal to mechanistic studies of proton translocation by membrane-bound enzymes. Here we report a detailed characterization of the pH-sensitive fluorescent nanoprobe Glu(3), which is well suited for pH measurements in microcompartmentalized biological systems. The probe is a polyglutamic porphyrin dendrimer in which multiple carboxylate termini ensure its high water solubility and prevent its diffusion across phospholipid membranes. The probe's pK is in the physiological pH range, and its protonation can be followed ratiometrically by absorbance or fluorescence in the ultraviolet-visible spectral region. The usefulness of the probe was enhanced by using a semiautomatic titration system coupled to a charge-coupled device (CCD) spectrometer, enabling fast and accurate titrations and full spectral coverage of the system at millisecond time resolution. The probe's pK was measured in bulk solutions as well as inside large unilamellar vesicles in the presence of physiologically relevant ions. Glu(3) was found to be completely membrane impermeable, and its distinct spectroscopic features permit pH measurements inside closed membrane vesicles, enabling quantitative mechanistic studies of membrane-spanning proteins. Performance of the probe was demonstrated by monitoring the rate of proton leakage through the phospholipid bilayer in large vesicles with and without the uncoupler gramicidin present. Overall, as a probe for biological proton translocation measurements, Glu(3) was found to be superior to the commercially available pH indicators.
Asunto(s)
Sondas Moleculares , Nanotecnología/métodos , Porfirinas/química , Liposomas Unilamelares/química , Concentración de Iones de Hidrógeno , Estructura Molecular , Ácido Poliglutámico/química , Espectrometría de FluorescenciaRESUMEN
The discovery that the native PS II enzyme undergoes charge separation via an absorption extending to 730 nm has led us to re-examine the low-temperature absorption spectra of Nanba-Satoh PS II reaction centre preparations with particular focus on the long wavelength region. It is shown that these preparations do not exhibit absorption in the 700-730 nm region at 1.7 K. Absorption in the Nanba-Satoh type preparations analogous to the 'red tail' as observed in functional PS II core complexes is likely shifted to higher energy by >20 nm. Spectral changes associated with the stable reduction of pheo(a) in chemically treated reaction centre preparations are also revisited. Dithionite treatment of PS II preparations in the dark leads to changes of pigment-pigment and/or pigment-protein interactions, as evidenced by changes in absorption and CD spectra. Absorption and CD changes associated with stable Pheo(D1) photo-reduction in PS II core complexes and Nanba-Satoh preparations are compared. For Nanba-Satoh preparations, Q(y) bleaches are approximately 3x broader than in PS II core complexes and are blue-shifted by approximately 4 nm. These data are discussed in terms of current models of PS II, and suggest a need to consider protein-induced changes of some electronic properties of reaction centre pigments.
Asunto(s)
Complejo de Proteína del Fotosistema II/química , Absorción , Dicroismo Circular , Frío , Oscuridad , Ditionita/química , Transporte de Electrón , Oxidación-Reducción , Fotosíntesis , Complejo de Proteína del Fotosistema II/metabolismo , Análisis EspectralRESUMEN
Low-temperature absorption and fluorescence spectra of fully active cores and membrane-bound PS II preparations are compared. Detailed temperature dependence of fluorescence spectra between 5 and 70 K are presented as well as 1.7-K fluorescence line-narrowed (FLN) spectra of cores, confirming that PS II emission is composite. Spectra are compared to those reported for LHCII, CP43, CP47 and D1/D2/cytit b559 subunits of PS II. A combination of subunit spectra cannot account for emission of active PS II. The complex temperature dependence of PS II fluorescence is interpretable by noting that excitation transfer from CP43 and CP47 to the reaction centre is slow, and strongly dependent on the precise energy at which a 'slow-transfer' pigment in CP43 or CP47 is located within its inhomogeneous distribution. PS II fluorescence arises from CP43 and CP47 'slow-transfer' states, convolved by this dependence. At higher temperatures, thermally activated excitation transfer to the PS II charge-separating system bypasses such bottlenecks. As the charge-separating state of active PS II absorbs at >700 nm, PS II emission in the 680-700 nm region is unlikely to arise from reaction centre pigments. PS II emission at physiological temperatures is discussed in terms of these results.
Asunto(s)
Fluorescencia , Oxígeno/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Spinacia oleracea/metabolismo , Temperatura , Transferencia de Energía , Spinacia oleracea/químicaRESUMEN
Active Photosystem II (PS II) cores were prepared from spinach, pea, Synechocystis PCC 6803, and Thermosynechococcus vulcanus, the latter of which has been structurally determined [Kamiya and Shen (2003) Proc Natl Acad Sci USA 100: 98-103]. Electrochromic shifts resulting from QA reduction by 1.7-K illumination were recorded, and the Qx and Qy absorption bands of the redox-active pheophytin a thus identified in the different organisms. The Qx transition is approximately 3 nm (100 cm-1) to higher energy in cyanobacteria than in the plants. The predominant Qy shift appears in the range 683-686 nm depending on species, and does not appear to have a systematic shift. Low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of the chlorophyll Qy region are very similar in spinach and pea, but vary in cyanobacteria. We assigned CP43 and CP47 trap-chlorophyll absorption features in all species, as well as a P680 transition. Each absorption identified has an area of one chlorophyll a. The MCD deficit, introduced previously for spinach as an indicator of P680 activity, occurs in the same spectral region and has the same area in all species, pointing to a robustness of this as a signature for P680. MCD and CD characteristics point towards a significant variance in P680 structure between cyanobacteria, thermophilic cyanobacteria, and higher plants.
Asunto(s)
Cianobacterias/química , Complejo de Proteína del Fotosistema II/química , Pisum sativum/química , Spinacia oleracea/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clorofila/química , Clorofila/metabolismo , Dicroismo Circular , Cianobacterias/metabolismo , Transferencia de Energía , Pisum sativum/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Especificidad de la Especie , Espectrofotometría Atómica , Spinacia oleracea/metabolismoRESUMEN
We report and compare highly resolved, simultaneously recorded absorption and CD spectra of active Photosystem II (PSII) samples in the range 440-750 nm. From an appropriately scaled comparison of spinach membrane fragment (BBY) and PSII core spectra, we show that key features of the core spectrum are quantitatively represented in the BBY data. PSII from the cyanobacterium Synechocystis 6803 display spectral features in the Qy region of comparable width (50-70 cm(-1) fwhm) to those seen in plant PSII but the energies of the resolved features are distinctly different. A comparison of spectra taken of PSII poised in the S1QA and S2QA(-) redox states reveals electrochromic shifts largely attributable to the influence of QA(-) on Pheo(D1). This allows accurate determinations of the Pheo(D1) Qy absorption positions to be at 685.0 nm for spinach cores, 685.8 nm for BBY particles, and 683.0 nm for Synechocystis. These are discussed in terms of earlier reports of the Pheo(D1) energies in PSII. The Qx transition of Pheo(D1) undergoes a blue shift upon Q(A) reduction, and we place a lower limit of 80 cm(-1) on this shift in plant material. By comparing the magnitude of the Stark shifts of the Qx and Qy bands of Pheo(D1), the directions of the transition-induced dipole moment changes, Deltamu(x) and Deltamu(y), for this functionally important pigment could be determined, assuming normal magnitudes of the Deltamu's. Consequently, Deltamu(x) and Deltamu(y) are determined to be approximately orthogonal to the directions expected for these transitions. Low-fluence illumination experiments at 1.7 K resulted in very efficient formation of QA(-). This was accompanied by cyt b(559) oxidation in BBYs and carotenoid oxidation in cores. No chlorophyll oxidation was observed. Our data allow us to estimate the quantum efficiency of PSII at this temperature to be of the order 0.1-1. No Stark shift associated with the S1-to-S2 transition of the Mn cluster is evident in our samples. The similarity of Stark data in plants and Synechocystis points to minimal interactions of Pheo(D1) with nearby chloropyll pigments in active PSII preparations. This appears to be at variance with interpretations of experiments performed with inactive solubilized reaction-center preparations.