Excited state charge-transfer dynamics study of poplar plastocyanin by ultrafast pump-probe spectroscopy and molecular dynamics simulation

We have applied ultrafast pump-probe spectroscopy to investigate the excited state dynamics of the blue copper protein poplar plastocyanin, by exciting in the blue side of its 600-nm absorption band. The decay of the charge-transfer excited state occurs exponentially with a time constant of approximately 280 fs and is modulated by well visible oscillations. The Fourier transform of the oscillatory component, besides providing most of the vibrational modes found by conventional resonance Raman, presents additional bands in the low frequency region modes, which are reminiscent of collective motions of biological relevance. Notably, a high frequency mode at approximately 508 cm(-1), whose dynamics are consistent with that of the excited state and already observed for other blue copper proteins, is shown to be present also in poplar plastocyanin. This vibrational mode is reproduced by a molecular dynamics simulation involving the excited state of the copper site.     

Time-resolved methods in biophysics. 4. Broadband pump-probe spectroscopy system with sub-20 fs temporal resolution for the study of energy transfer processes in photosynthesis.

In this paper we discuss how to push the temporal resolution limits of transient absorption spectroscopy in order to detect very fast processes (energy relaxation, energy or charge transfer, vibrational coherence) taking place in molecules of biological relevance. After reviewing the main principles of femtosecond pump-probe spectroscopy, we describe an experimental setup based on two synchronized non-collinear optical parametric amplifiers (NOPAs). Each NOPA can be independently configured to generate ultra-broadband sub-10 fs visible pulses, tunable 10-15 fs visible pulses, tunable 15-40 fs near-infrared pulses (900-1500 nm). This system enables to perform pump-probe experiments over nearly two octaves of spectrum with sub-20 fs temporal resolution. We then present an application example highlighting the capability of this instrument to track excited state dynamics in biomolecules on the sub-100 fs timescale: the study of carotenoid-bacteriochlorophyll energy transfer processes in peripheral light-harvesting complexes (LH2) from purple bacteria. We show that, by comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it is possible to visualize in the time domain the primary events in photosynthesis.     

Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes

Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum.     

Genetic heterogeneity in Italian families with IgA nephropathy: suggestive linkage for two novel IgA nephropathy loci

IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide, but its etiologic mechanisms are still poorly understood. Different prevalences among ethnic groups and familial aggregation, together with an increased familial risk, suggest important genetic influences on its pathogenesis. A locus for familial IgAN, called "IGAN1," on chromosome 6q22-23 has been described, without the identification of any responsible gene. The partners of the European IgAN Consortium organized a second genomewide scan in 22 new informative Italian multiplex families. A total of 186 subjects (59 affected and 127 unaffected) were genotyped and were included in a two-stage genomewide linkage analysis. The regions 4q26-31 and 17q12-22 exhibited the strongest evidence of linkage by nonparametric analysis (best P=.0025 and .0045, respectively). These localizations were also supported by multipoint parametric analysis, in which peak LOD scores of 1.83 ( alpha =0.50) and 2.56 ( alpha =0.65) were obtained using the affected-only dominant model, and by allowance for the presence of genetic heterogeneity. Our results provide further evidence for genetic heterogeneity among families with IgAN. Evidence of linkage to multiple chromosomal regions is consistent with both an oligo/polygenic and a multiple-susceptibility-gene model for familial IgAN, with small or moderate effects in determining the pathological phenotype. Although we identified new candidate regions, replication studies are required to confirm the genetic contribution to familial IgAN.     

Antiangiogenic Arming of an Oncolytic Vaccinia Virus Enhances Antitumor Efficacy in Renal Cell Cancer Models

Oncolytic vaccinia viruses have shown compelling results in preclinical cancer models and promising preliminary safety and antitumor activity in early clinical trials. However, to facilitate systemic application it would be useful to improve tumor targeting and antitumor efficacy further. Here we report the generation of vvdd-VEGFR-1-Ig, a targeted and armed oncolytic vaccinia virus. Tumor targeting was achieved by deletion of genes for thymidine kinase and vaccinia virus growth factor, which are necessary for replication in normal but not in cancer cells. Given the high vascularization typical of kidney cancers, we armed the virus with the soluble vascular endothelial growth factor (VEGF) receptor 1 protein for an antiangiogenic effect. Systemic application of high doses of vvdd-VEGFR-1-Ig resulted in cytokine induction in an immunocompromised mouse model. Upon histopathological analysis, splenic extramedullary hematopoiesis was seen in all virus-injected mice and was more pronounced in the vvdd-VEGFR-1-Ig group. Analysis of the innate immune response after intravenous virus injection revealed high transient and dose-dependent cytokine elevations. When medium and low doses were used for intratumoral or intravenous injection, vvdd-VEGFR-1-Ig exhibited a stronger antitumor effect than the unarmed control. Furthermore, expression of VEGFR-1-Ig was confirmed, and a concurrent antiangiogenic effect was seen. In an immunocompetent model, systemic vvdd-VEGFR-1-Ig exhibited superior antitumor efficacy compared to the unarmed control virus. In conclusion, the targeted and armed vvdd-VEGFR-1-Ig has promising anticancer activity in renal cell cancer models. Extramedullary hematopoiesis may be a sensitive indicator of vaccinia virus effects in mice.In 2002 renal cell cancer accounted for more than 200,000 cases and 100,000 deaths worldwide (33). Unfortunately, chemotherapy, radiotherapy, and immunotherapy yield low response rates (9, 17) in this cancer type. Thus, prognosis for patients is poor, especially when the disease is metastatic, as median survival is only 8 months (19). Although recently approved drugs, such as sorafenib, sunitinib, temsirolimus, and bevacizumab, have provided additional tools for treatment of renal cell cancer (7), they are usually not curative, and thus new treatment approaches are needed.Oncolytic vaccinia viruses are promising agents for cancer treatment and have shown compelling results in preclinical tumor models (40, 42, 45). Moreover, good safety and preliminary evidence of antitumor efficacy were seen in phase 1 clinical trials (22, 26, 32). Vaccinia virus has a strong oncolytic effect due to its fast replication cycle (45) and a high innate tropism to cancer tissue (34). Tumor targeting can be further improved by deleting vaccinia virus genes that are necessary for replication in normal cells but not in cancer cells. For example, deletions of either thymidine kinase (TK) or vaccinia virus growth factor (VGF) or both have been shown to reduce pathogenicity compared to wild-type virus (3, 5, 27). To enhance antitumor potency, oncolytic vaccinia viruses can be armed with therapeutic transgenes, such as immunostimulatory factors (26) or suicide genes (14, 16, 35). With regard to kidney cancer, an arming approach with antiangiogenenic molecules seems logical, considering the high vascularization characteristic of renal tumors (20).Vascular endothelial growth factor (VEGF) is a major player in tumor angiogenesis and is highly expressed in renal cell cancers (29). VEGF binds to the fms-like-tyrosine kinase receptor (flt-1 or VEGFR-1) and kinase domain region receptor (KDR or VEGFR-2) with high affinity (13). The soluble vascular endothelial growth factor receptor 1-Ig fusion protein (VEGFR-1-Ig) used in this study is derived from the membrane-bound VEGFR-1 and binds human and murine VEGF without inducing vascular endothelial cell mitogenesis (31). Blocking VEGF with this or closely related molecules has been shown to inhibit tumor growth in several cancer models (18, 21, 25, 39).Although tumor cell selective replication can be enhanced by deletion of TK and/or VGF to reduce pathogenicity (3, 5, 27), high doses of attenuated vaccinia virus may increase serum cytokine concentrations which parallel the onset of toxic events, as seen with other viral vectors (2, 38). The potential “early” toxicity associated with oncolytic vaccinia viruses has not been completely elucidated heretofore (36, 46).Given the high vascularization of renal cell cancers and the pressing need to generate new antitumor agents with increased safety and efficacy, we hypothesized that an oncolytic vaccinia virus targeted by TK and VGF deletions and armed with VEGFR-1-Ig would exhibit enhanced antitumor efficacy due to its antiangiogenic properties in renal cell cancer models compared to a nonarmed control virus, allowing reduction of the treatment dose.     

Carotenoid-bacteriochlorophyll energy transfer in LH2 complexes studied with 10-fs time resolution

In this report, we present a study of carotenoid-bacteriochlorophyll energy transfer processes in two peripheral light-harvesting complexes (known as LH2) from purple bacteria. We use transient absorption spectroscopy with approximately 10 fs temporal resolution, which is necessary to observe the very fast energy relaxation processes. By comparing excited-state dynamics of the carotenoids in organic solvents and inside the LH2 complexes, it has been possible to directly evaluate their energy transfer efficiency to the bacteriochlorophylls. In the case of okenone in the LH2 complex from Chromatium purpuratum, we obtained an energy transfer efficiency of etaET2=63+/-2.5% from the optically active excited state (S2) and etaET1=61+/-2% from the optically dark state (S1); for rhodopin glucoside contained in the LH2 complex from Rhodopseudomonas acidophila these values become etaET2=49.5+/-3.5% and etaET1=5.1+/-1%. The measurements also enabled us to observe vibrational energy relaxation in the carotenoids' S1 state and real-time collective vibrational coherence initiated by the ultrashort pump pulses. Our results are important for understanding the dynamics of early events of photosynthesis and relating it to the structural arrangement of the chromophores.     

Effects of tunable excitation in carotenoids explained by the vibrational energy relaxation approach

Carotenoids are fundamental building blocks of natural light harvesters with convoluted and ultrafast energy deactivation networks. In order to disentangle such complex relaxation dynamics, several studies focused on transient absorption measurements and their dependence on the pump wavelength. However, such findings are inconclusive and sometimes contradictory. In this study, we compare internal conversion dynamics in \(\beta\)-carotene, pumped at the first, second, and third vibronic progression peak. Instead of employing data fitting algorithms based on global analysis of the transient absorption spectra, we apply a fully quantum mechanical model to treat the high-frequency symmetric carbon–carbon (C=C and C–C) stretching modes explicitly. This model successfully describes observed population dynamics as well as spectral line shapes in their time-dependence and allows us to reach two conclusions: Firstly, the broadening of the induced absorption upon excess excitation is an effect of vibrational cooling in the first excited state (\(S_{1}\)). Secondly, the internal conversion rate between the second excited state (\(S_{2}\)) and \(S_{1}\) crucially depends on the relative curve displacement. The latter point serves as a new perspective on solvent- and excitation wavelength-dependent experiments and lifts contradictions between several studies found in literature.     

Carotenoid-to-bacteriochlorophyll energy transfer through vibronic coupling in LH2 from <Emphasis Type="Italic">Phaeosprillum molischianum</Emphasis>

The peripheral light-harvesting antenna complex (LH2) of purple photosynthetic bacteria is an ideal testing ground for models of structure–function relationships due to its well-determined molecular structure and ultrafast energy deactivation. It has been the target for numerous studies in both theory and ultrafast spectroscopy; nevertheless, certain aspects of the convoluted relaxation network of LH2 lack a satisfactory explanation by conventional theories. For example, the initial carotenoid-to-bacteriochlorophyll energy transfer step necessary on visible light excitation was long considered to follow the Förster mechanism, even though transfer times as short as 40 femtoseconds (fs) have been observed. Such transfer times are hard to accommodate by Förster theory, as the moderate coupling strengths found in LH2 suggest much slower transfer within this framework. In this study, we investigate LH2 from Phaeospirillum (Ph.) molischianum in two types of transient absorption experiments—with narrowband pump and white-light probe resulting in 100 fs time resolution, and with degenerate broadband 10 fs pump and probe pulses. With regard to the split Q_x band in this system, we show that vibronically mediated transfer explains both the ultrafast carotenoid-to-B850 transfer, and the almost complete lack of transfer to B800. These results are beyond Förster theory, which predicts an almost equal partition between the two channels.