Spectral Hole Burning in Cyanobacterial Photosystem I with P700 in Oxidized and Neutral States.

We explored the rich satellite hole structures emerging as a result of spectral hole burning in cyanobacterial photosystem I (PSI) and demonstrated that hole burning properties of PSI, particularly at high resolution, are strongly affected by the oxidation state of the primary donor P700, as P700+ effectively quenches the excitations of the lowest-energy antenna states responsible for fluorescence. Obtaining better control of this variable will be crucial for high-resolution ensemble experiments on protein energy landscapes in PSI. The separate nonphotochemical spectral hole burning (NPHB) signatures of various red antenna states were obtained, allowing for additional constraints on excitonic structure-based calculations. Preliminary evidence is presented for an additional red state of PSI of T. elongatus peaked at 712.6 nm, distinct from previously reported C708 and C715 states and possibly involving chlorophyll B15. Excitation at wavelengths as long as 800 nm results in charge separation at cryogenic temperatures in PSI also in Synechocystis sp. PCC 6803. Both the "P700+ minus P700" holes and nonphotochemical spectral holes were subjected to thermocycling. The distribution of barriers manifesting in recovery of the "P700+ minus P700" signature contains two components in sample-dependent proportions, likely reflecting the percentages of FA and FB clusters being successfully prereduced before the optical experiment. The barrier distribution for the recovery of the lower-energy nonphotochemical spectral holes resembles those observed for other pigment-protein complexes, suggesting similar structural elements are responsible for NPHB. Higher-energy components exhibit evidence of "domino effects" such as shifts of certain bands persisting past the lower-energy hole recovery. Thus, conformational changes triggered by excitation of one pigment likely can affect multiple pigments in this tightly packed system.

Conformational Changes in Pigment-Protein Complexes at Low Temperatures-Spectral Memory and a Possibility of Cooperative Effects.

We employed nonphotochemical hole burning (NPHB) and fluorescence line narrowing (FLN) spectroscopies to explore protein energy landscapes and energy transfer processes in dimeric Cytochrome b6f, containing one chlorophyll molecule per protein monomer. The parameters of the energy landscape barrier distributions quantitatively agree with those reported for other pigment-protein complexes involved in photosynthesis. Qualitatively, the distributions of barriers between protein substates involved in the light-induced conformational changes (i.e., -NPHB) are close to glass-like ∼1/√V (V is the barrier height) and not to Gaussian. There is a high degree of correlation between the heights of the barriers in the ground and excited states in individual pigment-protein systems, as well as nearly perfect spectral memory. Both NPHB and hole recovery are due to phonon-assisted tunneling associated with the increase of the energy of a scattered phonon. As the latter is unlikely for simultaneously both the hole burning and the hole recovery, proteins must exhibit a NPHB mechanism involving diffusion of the free volume toward the pigment. Entities involved in the light-induced conformational changes are characterized by md(2) value of about 1.0 × 10(-46) kg·m(2). Thus, these entities are protons or, alternatively, small groups of atoms experiencing sub-Å shifts. However, explaining all spectral hole burning and recovery data simultaneously, employing just one barrier distribution, requires a drastic decrease in the attempt frequency to about 100 MHz. This decrease may occur due to cooperative effects. Evidence is presented for excitation energy transfer between the chlorophyll molecules of the adjacent monomers. The magnitude of the dipole-dipole coupling deduced from the Δ-FLN spectra is in good agreement with the structural data, indicating that the explored protein was intact.

Modeling of various optical spectra in the presence of slow excitation energy transfer in dimers and trimers with weak interpigment coupling: FMO as an example.

We present an improved simulation methodology to describe nonphotochemical hole-burned (NPHB) spectra. The model, which includes both frequency-dependent excitation energy transfer (EET) rate distributions and burning following EET, provides reasonable fits of various optical spectra including resonant and nonresonant holes in the case of FMO complex. A qualitative description of the NPHB process in light of a very complex protein energy landscape is briefly discussed. As an example, we show that both resonant and nonresonant HB spectra obtained for the 825 nm band of the trimeric FMO of C. tepidum are consistent with the presence of a relatively slow EET between the lowest energy states of the monomers of the trimer (mostly localized on BChl a 3), with a weak (∼1 cm(-1)) coupling between these states revealed via calculated emission spectra. We argue that the nature of the so-called 825 nm absorption band of the FMO trimer, contrary to the presently accepted consensus, cannot be explained by a single transition.

Spectral hole burning, recovery, and thermocycling in chlorophyll-protein complexes: distributions of barriers on the protein energy landscape.

Chlorophyll-protein complexes are ideal model systems for protein energy landscape research. Here pigments, used in optical spectroscopy experiments as sensitive probes to local dynamics, are built into protein by Nature (in a large variety of local environments; without extraneous chemical manipulations or genetic engineering). Distributions of the tunneling parameter, λ, and/or protein energy landscape barrier heights, V, have been determined for (the lowest energy state of) the CP43 core antenna complex of photosystem II. We demonstrate that spectral hole burning (SHB) and hole recovery (HR) measurements are capable of delivering important information on protein energy landscape properties and spectral diffusion mechanism details. In particular, we show that tunneling rather than barrier hopping is responsible for both persistent SHB and subsequent HR at 5-12 K, which allows us to estimate the md(2) parameter of the tunneling entities as ~1.0 × 10(-46) kg·m(2). The subdistributions of λ actually contributing to the nonsaturated spectral holes (and affecting their recovery) differ from the respective full true distributions. In the case of the full λ-distribution being uniform (or the barrier height distribution ~1/√V, a model which has been widely employed in theories of amorphous solids at low temperatures and in HR analysis), the difference is qualitative, with λ subdistributions probed in the HR experiments being highly asymmetrical, and barrier V subdistributions deviating significantly from ~1/√V. Thus, the distribution of λ for the protein energy landscape tier directly probed by SHB is likely Gaussian and not uniform. Additionally, a Gaussian distribution of barriers, with parameters incompatible with those of the landscape tier directly probed by SHB, contributes to the thermocycling results.

Effects of the distributions of energy or charge transfer rates on spectral hole burning in pigment-protein complexes at low temperatures.

Effects of the distributions of excitation energy transfer (EET) rates (homogeneous line widths) on the nonphotochemical (resonant) spectral hole burning (SHB) processes in photosynthetic chlorophyll-protein complexes (reaction center [RC] and CP43 antenna of Photosystem II from spinach) are considered. It is demonstrated that inclusion of such a distribution results in somewhat more dispersive hole burning kinetics. More importantly, however, inclusion of the EET rate distributions strongly affects the dependence of the hole width on the fractional hole depth. Different types of line width distributions have been explored, including those resulting from Förster type EET between weakly interacting pigments as well as Gaussian ones, which may be a reasonable approximation for those resulting, for instance, from so-called extended Förster models. For Gaussian line width distributions, it is possible to determine the parameters of both line width and tunneling parameter distributions from SHB data without a priori knowledge of any of them. Concerning more realistic asymmetric distributions, we demonstrate, using the simple example of CP43 antenna, that one can use SHB modeling to estimate electrostatic couplings between pigments and support or exclude assignment of certain pigment(s) to a particular state.

Parameters of the protein energy landscapes of several light-harvesting complexes probed via spectral hole growth kinetics measurements.

The parameters of barrier distributions on the protein energy landscape in the excited electronic state of the pigment/protein system have been determined by means of spectral hole burning for the lowest-energy pigments of CP43 core antenna complex and CP29 minor antenna complex of spinach Photosystem II (PS II) as well as of trimeric and monomeric LHCII complexes transiently associated with the pea Photosystem I (PS I) pool. All of these complexes exhibit sixty to several hundred times lower spectral hole burning yields as compared with molecular glassy solids previously probed by means of the hole growth kinetics measurements. Therefore, the entities (groups of atoms), which participate in conformational changes in protein, appear to be significantly larger and heavier than those in molecular glasses. No evidence of a small (∼1 cm(-1)) spectral shift tier of the spectral diffusion dynamics has been observed. Therefore, our data most likely reflect the true barrier distributions of the intact protein and not those related to the interface or surrounding host. Possible applications of the barrier distributions as well as the assignments of low-energy states of CP29 and LHCII are discussed in light of the above results.

Low-temperature protein dynamics of the B800 molecules in the LH2 light-harvesting complex: spectral hole burning study and comparison with single photosynthetic complex spectroscopy.

Previously published and new spectral hole burning (SHB) data on the B800 band of LH2 light-harvesting antenna complex of Rps. acidophila are analyzed in light of recent single photosynthetic complex spectroscopy (SPCS) results (for a review, see Berlin et al. Phys. Life Rev. 2007, 4, 64.). It is demonstrated that, in general, SHB-related phenomena observed for the B800 band are in qualitative agreement with the SPCS data and the protein models involving multiwell multitier protein energy landscapes. Regarding the quantitative agreement, we argue that the single-molecule behavior associated with the fastest spectral diffusion (smallest barrier) tier of the protein energy landscape is inconsistent with the SHB data. The latter discrepancy can be attributed to SPCS probing not only the dynamics of of the protein complex per se, but also that of the surrounding amorphous host and/or of the host-protein interface. It is argued that SHB (once improved models are developed) should also be able to provide the average magnitudes and probability distributions of light-induced spectral shifts and could be used to determine whether SPCS probes a set of protein complexes that are both intact and statistically relevant. SHB results are consistent with the B800 --> B850 energy-transfer models including consideration of the whole B850 density of states.

Photodynamic therapy of Walker tumors by multiple laser irradiation.

OBJECTIVE: Photodynamic therapy of Walker tumor after subcutaneous administration of 5-ALA solution using a multiple laser irradiation scheme was monitored by the fluorescence imaging technique to investigate the effectiveness of 5-ALA-PDT. BACKGROUND DATA: Photodynamic therapy (PDT) is a localized cancer treatment based on the selective uptake and retention of photosensitizer at the tumoral level and on the activation of the photosensitizer by a specific wavelength of light, aiming to induce cytotoxic reactions. As a new photosensitizer, the heme precursor 5- aminolevulinic acid has been introduced recently for photodynamic therapy of tumors and precancerous lesions of the skin. It has been shown that the efficacy of topical 5-ALA-PDT is limited for deeper skin tumor by the depth of 5-ALA penetration through the skin. Oral or systemic administration of ALA or the use of different irradiation schemes may improve tumor response to PDT. METHODS: Laser irradiation parameters used in this study were lambda = 635 nm, P = 3 mW, t(exp) = 300 sec, and three sessions. The fluorescence was excitated by monochromatic light of 405 nm. The temporal behavior of PpIX fluorescence was studied by processing and analyzing the fluorescence images acquired just after applying 5-ALA, just before and just after three laser irradiations. RESULTS: The results demonstrate that PpIX is highly selective for tumors areas and a re-accumulation of PpIX appears between laser irradiations. During laser irradiation, the PpIX fluorescence intensity decreases rapidly, reflecting the photodegradation of PpIX. CONCLUSIONS: In conclusion, the use of a multiple laser irradiation scheme, for the activation of reaccumulation of Pp IX (with three steps) is effective for photodynamic therapy of Walker tumor.

Low-level laser therapy (LLLT) efficacy in post-operative wounds.

OBJECTIVE: The aim of this paper was to investigate the efficacy of low-level laser radiation (LLLR) with wavelength of 904 nm on the stimulation of the healing process of postoperative aseptic wounds (early scar). BACKGROUND DATA: Low-level laser therapy (LLLT) has been increasingly used to treat many disorders, including wounds. However, despite such increased clinical usage, there is still controversy regarding the efficacy of this wound treatment in curent clinical practice. METHODS: LLLT has been used to treat cutting plague in the right instep and on the left foot. Both resulted from sutured wounds. The clinical evaluation by semiquantitative methods is presented. RESULTS: Clinical evaluation showed that the healing process of these postoperatively treated wounds has occurred and that the functional recovery of the patients (i.e., return to their ordinary life) was faster than without treatment. CONCLUSION: LLLR with wavelength of 904 nm to stimulate postoperative aseptic wounds (early scar) is efficient in both cases of cutting plague.

The monitoring of the accumulation of protoporphyrin IX in Walker tumours by subcutaneous administration of delta-aminolevulinic acid.

Photodynamic therapy with protoporphyrin IX induced by delta-aminolevulinic acid (ALA) is mainly applied for the treatment of human superficial skin cancer. In this paper we present our study on photodynamic therapy (PDT) of the implanted Walker tumours using subcutaneous administration of ALA to improve the availability of ALA in the skin. We determined the accumulation and localization of protoporphyrin IX (PpIX) after subcutaneous administration of different concentrations of ALA in a physiological saline solutions, using fluorescence imaging technique. The results obtained indicate that PpIX accumulation depends on the concentration of ALA. The temporal behavior of PpIX fluorescence has shown a clear demarcation of tumoural zone depending on the post-administration time and the administrated concentration of the ALA solution. Further studies are needed to confirm these encouraging results and to define the PDT protocols using subcutaneous administration of ALA solution