Results of directly applied activated carbon cloth in chronic wounds: a preliminary study.

OBJECTIVE: Activated carbon (AC) has been used in wound therapy as an active substance inside dressings. Applying AC directly on a wound is a new concept. The aim of this study was to analyse the outcomes of chronic wounds which were managed with directly applied activated carbon knitted cloth (ACC, Zorflex) in Swiss patients. METHOD: A retrospective analysis of the records of all patients with chronic wounds treated with ACC between 1 October 2013 and 31 December 2015 in an outpatient wound clinic. Chronic was defined as a wound being present for >3 weeks. Malignant wounds were excluded. The main outcome was the time to complete closure or readiness for spilt-thickness skin grafting (STSG). Descriptive data, including nutritional status and angiology results were obtained. RESULTS: There were 36 women and 34 men, median age 68 years old. The median body mass index (BMI) 28.1kg/m2 and 76% (n=53) of patients had comorbidities. Angiology exam results showed signs of reduced arterial perfusion in 13% (n=9) of patients and malnutrition in 11% (n=8). Of the wounds included 34% (n=24) were on the trunk and 66% (n=46) on the extremities. The median wound size was 6.9cm2 (range: 0.1-300cm2). The wounds on the trunk were larger than wounds on extremities (10 versus 2cm2). Overall, median time to wound closure was 51 days. In 94% (n=66) of patients, wounds closed without further intervention and 6% (n=4) underwent STSG. Patients with comorbidities showed longer wound healing times compared with those without. No adverse events such as allergies or skin irritation occurred. Cost analysis, including personnel and material and stratified according known wound closure times, showed ACC (US$ 1252) to be like hydrocolloids (US$ 1128), but substantially lower than white gauze (US$ 3026) and negative pressure wound therapy (NPWT) (US$ 2578). CONCLUSION: ACC applied directly on chronic wounds of different aetiology is safe with short closure times. The cost efficiency is high. It combines the positive features of other wound dressings, such as hydrocolloids and NPWT, without their disadvantages. The dressing change of ACC is easy and non-specialised nurses or even patients themselves can be taught to perform it.

Synapse fits neuron: joint reduction by model inversion.

In this paper, we introduce a novel simplification method for dealing with physical systems that can be thought to consist of two subsystems connected in series, such as a neuron and a synapse. The aim of our method is to help find a simple, yet convincing model of the full cascade-connected system, assuming that a satisfactory model of one of the subsystems, e.g., the neuron, is already given. Our method allows us to validate a candidate model of the full cascade against data at a finer scale. In our main example, we apply our method to part of the squid's giant fiber system. We first postulate a simple, hypothetical model of cell-to-cell signaling based on the squid's escape response. Then, given a FitzHugh-type neuron model, we derive the verifiable model of the squid giant synapse that this hypothesis implies. We show that the derived synapse model accurately reproduces synaptic recordings, hence lending support to the postulated, simple model of cell-to-cell signaling, which thus, in turn, can be used as a basic building block for network models.

Preserved oxygenation in obese patients receiving protective ventilation during laparoscopic surgery: a randomized controlled study.

BACKGROUND: Venous admixture from atelectasis and airway closure impedes oxygenation during general anaesthesia. We tested the hypothesis that continuous positive airway pressure (CPAP) during pre-oxygenation and reduced fraction of inspiratory oxygen (FIO2 ) during emergence from anaesthesia can improve oxygenation in patients with obesity undergoing laparoscopic surgery. METHODS: In the intervention group (n = 20, median BMI 41.9), a CPAP of 10 cmH2 O was used during pre-oxygenation and induction of anaesthesia, but no CPAP was used in the control group (n = 20, median BMI 38.1). During anaesthesia, all patients were ventilated in volume-controlled mode with an FIO2 of 0.4 and a positive end-expiratory pressure (PEEP) of 10 cmH2 O. During emergence, before extubation, the control group was given an FIO2 of 1.0 and the intervention group was divided into two subgroups, which were given an FIO2 of 1.0 or 0.31. Oxygenation was assessed perioperatively by the estimated venous admixture (EVA). RESULTS: The median EVA before pre-oxygenation was about 8% in both groups. During anaesthesia after intubation, the median EVA was 8.2% in the intervention vs. 13.2% in the control group (P = 0.048). After CO2 pneumoperitoneum, the median EVA was 8.4% in the intervention vs. 9.9% in the control group (P > 0.05). One hour post-operatively, oxygenation had deteriorated in patients given an FIO2 of 1.0 during emergence but not in patients given an FIO2 of 0.31. CONCLUSIONS: A CPAP of 10 cmH2 O during pre-oxygenation and induction, followed by PEEP after intubation, seemed to preserve oxygenation during anaesthesia. Post-operative oxygenation depended on the FIO2 used during emergence.

Ultra-low-noise EEG/MEG systems enable bimodal non-invasive detection of spike-like human somatosensory evoked responses at 1 kHz.

Non-invasive EEG detection of very high frequency somatosensory evoked potentials featuring frequencies up to and above 1 kHz has been recently reported. Here, we establish the detectability of such components by combined low-noise EEG/MEG. We recorded SEP/SEF simultaneously using median nerve stimulation in five healthy human subjects inside an electromagnetically shielded room, combining a low-noise EEG custom-made amplifier (4.7 nV/√Hz) and a custom-made single-channel low-noise MEG (0.5 fT/√Hz @ 1 kHz). Both, low-noise EEG and MEG revealed three spectrally distinct and temporally overlapping evoked components: N20 (<100 Hz), sigma-burst (450-750 Hz), and kappa-burst (850-1200 Hz). The two recording modalities showed similar relative scaling of signal amplitude in all three frequencies domains (EEG [10 nV] ≅ MEG [1 fT]). Pronounced waveform (peak-by-peak) overlap of EEG and MEG signals is observed in the sigma band, whereas in the kappa band overlap was only partial. A decreasing signal-to-noise ratio (SNR; calculated for n = 12.000 averages) from sigma to kappa components characterizes both, electric and magnetic field recordings: Sigma-band SNR was 12.9  ±  5.5/19.8  ±  12.6 for EEG/MEG, and kappa-band SNR at 3.77  ±  0.8/4.5  ±  2.9. High-frequency performance of a tailor-made MEG matches closely with simultaneously recorded low-noise EEG for the non-invasive detection of somatosensory evoked activity at and above 1 kHz. Thus, future multi-channel dual-mode low-noise technology could offer complementary views for source reconstruction of the neural generators underlying such high-frequency responses, and render neural high-frequency processes related to multi-unit spike discharges accessible in non-invasive recordings.

Distinction between added-energy and phase-resetting mechanisms in non-invasively detected somatosensory evoked responses.

Non-invasively recorded averaged event-related potentials (ERP) represent a convenient opportunity to investigate human brain perceptive and cognitive processes. Nevertheless, generative ERP mechanisms are still debated. Two previous approaches have been contested in the past: the added-energy model in which the response raises independently from the ongoing background activity, and the phase-reset model, based on stimulus-driven synchronization of oscillatory ongoing activity. Many criteria for the distinction of these two models have been proposed, but there is no definitive methodology to disentangle them, owing also to the limited information at the single trial level. Here, we propose a new approach combining low-noise EEG technology and multivariate decomposition techniques. We present theoretical analyses based on simulated data and identify in high-frequency somatosensory evoked responses an optimal target for the distinction between the two mechanisms.

Towards non-invasive multi-unit spike recordings: mapping 1kHz EEG signals over human somatosensory cortex.

OBJECTIVE: Scalp-derived human somatosensory evoked potentials (SEPs) contain high-frequency oscillations (600 Hz; 'sigma-burst') reflecting concomitant bursts of spike responses in primary somatosensory cortex that repeat regularly at 600 Hz. Notably, recent human intracranial SEP have revealed also 1 kHz responses ('kappa-burst'), possibly reflecting non-rhythmic spiking summed over multiple cells (MUA: multi-unit activity). However, the non-invasive detection of EEG signals at 1 kHz typical for spikes has always been limited by noise contributions from both, amplifier and body/electrode interface. Accordingly, we developed a low-noise recording set-up optimised to map non-invasively 1 kHz SEP components. METHODS: SEP were recorded upon 4 Hz left median nerve stimulation in 6 healthy human subjects. Scalp potentials were acquired inside an electrically and magnetically shielded room using low-noise custom-made amplifiers. Furthermore, in order to reduce thermal Johnson noise contributions from the sensor/skin interface, electrode impedances were adjusted to ≤ 1 kΩ. Responses averaged after repeated presentation of the stimulus (n=4000 trials) were evaluated by spatio-temporal pattern analyses in complementary spectral bands. RESULTS: Three distinct spectral components were identified: N20 (<100 Hz), sigma-burst (450-750 Hz), and kappa-burst (850-1200 Hz). The two high-frequency bursts (sigma, kappa) exhibited distinct and partially independent spatiotemporal evolutions, indicating subcortical as well as several cortical generators. CONCLUSIONS: Using a dedicated low-noise set-up, human SEP 'kappa-bursts' at 1 kHz can be non-invasively detected and their scalp distribution be mapped. Their topographies indicate a set of subcortical/cortical generators, at least partially distinct from the topography of the 600 Hz sigma-bursts described previously. SIGNIFICANCE: The non-invasive detection and surface mapping of 1 kHz EEG signals presented here provides an essential step towards non-invasive monitoring of multi-unit spike activity.

Extension of non-invasive EEG into the kHz range for evoked thalamocortical activity by means of very low noise amplifiers.

Ultrafast electroencephalographic signals, having frequencies above 500 Hz, can be observed in somatosensory evoked potential measurements. Usually, these recordings have a poor signal-to-noise ratio (SNR) because weak signals are overlaid by intrinsic noise of much higher amplitude like that generated by biological sources and the amplifier. As an example, recordings at the scalp taken during electrical stimulation of the median nerve show a 600 Hz burst with submicro-volt amplitudes which can be extracted from noise by the use of massive averaging and digital signal processing only. We have investigated this signal by means of a very low noise amplifier made in-house (minimal voltage noise 2.7 nV Hz(-1/2), FET inputs). We examined how the SNR of the data is altered by the bandwidth and the use of amplifiers with different intrinsic amplifier noise levels of 12 and 4.8 nV Hz(-1/2), respectively. By analyzing different frequency contributions of the signal, we found an extremely weak 1 kHz component superimposed onto the well-known 600 Hz burst. Previously such high-frequency electroencephalogram responses around 1 kHz have only been observed by deep brain electrodes implanted for tremor therapy of Parkinson patients. For the non-invasive measurement of such signals, we recommend that amplifier noise should not exceed 4 nV Hz(-1/2).

Dynamical transition of protein-hydration water.

Thin layers of water on biomolecular and other nanostructured surfaces can be supercooled to temperatures not accessible with bulk water. Chen et al. [Proc. Natl. Acad. Sci. U.S.A. 103, 9012 (2006)]10.1073/pnas.0602474103 suggested that anomalies near 220 K observed by quasielastic neutron scattering can be explained by a hidden critical point of bulk water. Based on more sensitive measurements of water on perdeuterated phycocyanin, using the new neutron backscattering spectrometer SPHERES, and an improved data analysis, we present results that show no sign of such a fragile-to-strong transition. The inflection of the elastic intensity at 220 K has a dynamic origin that is compatible with a calorimetric glass transition at 170 K. The temperature dependence of the relaxation times is highly sensitive to data evaluation; it can be brought into perfect agreement with the results of other techniques, without any anomaly.

Stratum corneum lipids, skin barrier function and filaggrin mutations in patients with atopic eczema.

BACKGROUND: Prior to the discovery of filaggrin (FLG) mutations, evidence for an impaired skin barrier in atopic dermatitis (AD) has been documented, and changes in ceramide profile, altered skin pH and increased trans-epidermal water loss (TEWL) in patients with AD have been reported. Until now, no studies have analysed stratum corneum (SC) lipids combined with skin barrier parameters in subjects of known FLG genotype. METHODS: A cohort of 49 German individuals genotyped for the most common FLG mutations (R501X, 2282del4) had SC samples taken for lipid analysis by high-performance thin layer chromatography. In addition, TEWL, erythema, skin hydration and pH were measured. In 27 of the 49 individuals, a 24-h irritation patch test with sodium lauryl sulphate was performed. For the analysis, both the AD group and the control group were stratified by FLG mutation status (FLGmut/FLGwt). RESULTS: In the FLGmut AD group, significantly lower levels of ceramide 4 and significantly higher levels of ceramide 7 were observed when compared to both healthy control groups. However, ceramide 7 levels also significantly differed between FLGwt AD and FLGwt controls, as did ceramide 1 levels. No significant differences were observed for ceramide 2, 3, 5 and 6. FLGmut individuals had significantly higher skin pH values than individuals not carrying FLG mutations. Patients with AD with FLG mutations had significantly higher erythema compared to patients with AD without FLG mutations. CONCLUSION: Our results confirm previous observations of altered ceramide levels in AD, which however appear to show no clear relationship with FLG mutations.

Biliprotein maturation: the chromophore attachment.

Biliproteins are a widespread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. Covalent binding stabilizes the chromoproteins and is mandatory for phycobilisome assembly; and, it is also important in biliprotein applications such as fluorescence labelling. Covalent binding has, on the other hand, also considerably hindered biliprotein research because autocatalytic chromophore additions are rare, and information on enzymatic addition by lyases was limited to a single example, an EF-type lyase attaching phycocyanobilin to cysteine-alpha84 of C-phycocyanin. The discovery of new activities for the latter lyases, and of new types of lyases, have reinvigorated research activities in the subject. So far, work has mainly concentrated on cyanobacterial phycobiliproteins. Methodological advances in the process, however, as well as the finding of often large numbers of homologues, opens new possibilities for research on the subsequent assembly/disassembly of the phycobilisome in cyanobacteria and red algae, on the assembly and organization of the cryptophyte light-harvesting system, on applications in basic research such as protein folding, and on the use of phycobiliproteins for labelling.

Fluorescence spectroscopy of reconstituted peridinin-chlorophyll-protein complexes.

Peridinin-chlorophyll-proteins (PCP) were reconstituted with binary 1:1 chlorophyll (Chl) mixtures of Chl a, Chl b, [3-acetyl]-Chl a (acChl a), and studied by bulk and single-molecule fluorescence spectroscopy. The latter provides a way to distinguish in a given sample hetero-chlorophyllous complexes that contain two different Chls from homo-chlorophyllous ones containing the same Chl in both binding sites. The results are compared with those of homo-chlorophyllous PCP reconstituted with pure Chl a, Chl b, or acChl a. Relative intensities of the Chl fluorescence in hetero-chlorophyllous complexes were obtained and modeled using the Förster description of energy transfer combined with known variations of peridinin (Per)-Chl excitation transfer rates for the different Chl pigments. In the case of hetero-chlorophyllous complexes containing acChl a, the energy transfer is unidirectional in the energetically preferable direction, while it is bi-directional in the sample reconstituted with Chl a and Chl b.

Relative binding affinities of chlorophylls in peridinin-chlorophyll-protein reconstituted with heterochlorophyllous mixtures.

Peridinin-chlorophyll-protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these "half-mers", homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.

Monitoring fluorescence of individual chromophores in peridinin-chlorophyll-protein complex using single molecule spectroscopy.

Single molecule spectroscopy experiments are reported for native peridinin-chlorophyll a-protein (PCP) complexes, and three reconstituted light-harvesting systems, where an N-terminal construct of native PCP from Amphidinium carterae has been reconstituted with chlorophyll (Chl) mixtures: with Chl a, with Chl b and with both Chl a and Chl b. Using laser excitation into peridinin (Per) absorption band we take advantage of sub-picosecond energy transfer from Per to Chl that is order of magnitude faster than the Förster energy transfer between the Chl molecules to independently populate each Chl in the complex. The results indicate that reconstituted PCP complexes contain only two Chl molecules, so that they are spectroscopically equivalent to monomers of native-trimeric-PCP and do not aggregate further. Through removal of ensemble averaging we are able to observe for single reconstituted PCP complexes two clear steps in fluorescence intensity timetraces attributed to subsequent bleaching of the two Chl molecules. Importantly, the bleaching of the first Chl affects neither the energy nor the intensity of the emission of the second one. Since in strongly interacting systems Chl is a very efficient quencher of the fluorescence, this behavior implies that the two fluorescing Chls within a PCP monomer interact very weakly with each other which makes it possible to independently monitor the fluorescence of each individual chromophore in the complex. We apply this property, which distinguishes PCP from other light-harvesting systems, to measure the distribution of the energy splitting between two chemically identical Chl a molecules contained in the PCP monomer that reaches 280 cm(-1). In agreement with this interpretation, stepwise bleaching of fluorescence is also observed for native PCP complexes, which contain six Chls. Most PCP complexes reconstituted with both Chl a and Chl b show two emission lines, whose wavelengths correspond to the fluorescence of Chl a and Chl b. This is a clear proof that these two different chromophores are present in a single PCP monomer. Single molecule fluorescence studies of PCP complexes, both native and artificially reconstituted with chlorophyll mixtures, provide new and detailed information necessary to fully understand the energy transfer in this unique light-harvesting system.

Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: preparation, bulk and single molecule spectroscopy.

Reconstitution of the 16 kDa N-terminal domain of the peridinin-chlorophyll-protein, N-PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N-PCP. Besides homo-chlorophyllous complexes, hetero-chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo- and hetero-chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.

Effects of carotenoid inhibition on the photosynthetic RC-LH1 complex in purple sulphur bacterium Thiorhodospira sibirica.

Core complexes (LH1-RC) were isolated using preparative gel electrophoresis from photosynthetic membranes of the purple bacterium, Thiorhodospira sibirica, grown in the absence or presence of the carotenoid biosynthesis inhibitor, diphenylamine. The biosynthesis of carotenoids is affected by diphenylamine both quantitavely and qualitatively: after inhibition, the level of carotenoids in core complexes reaches only 10% of the normal content, as analyzed by HPLC and absorption spectroscopy. The normally grown bacterium biosynthesizes spirilloxanthin, rhodopin, anhydrorhodovibrin and lycopene, whereas after inhibition only neurosporene, zeta-carotene and their derivatives are found in the complexes. There is no concomitant accumulation of appreciable amounts of colorless carotenoid precursors. Interestingly, the main absorption band of the core light harvesting complex isolated from carotenoid-inhibited cells, shows a red shift to 889 nm, instead of a blue shift observed in many carotenoid-deficient species of purple photosynthetic bacteria. The stability of isolated core complexes against n-octyl-beta-D: -glucopyranoside clearly depends on the presence of carotenoids. Subcomplexes resulting from the detergent treatment, were characterized by non-denaturating gel electrophoresis combined with in situ absorption spectroscopy. Core complexes with the native carotenoid complement dissociate into three subcomplexes: (a) LH1 complexes partially depleted of carotenoids, with an unusual spectrum in the NIR region (lambdamax = 791, 818, 847 and 875 nm), (b) reaction centers associated with fragments of LH1, (c) small amounts of a carotenoidless B820 subcomplex. The core complex from the carotenoid-deficient bacterium is much less stable and yields only the two sub-complexes (b) and (c). We conclude that carotenoids contribute critically to stability and interactions of the core complexes with detergents.

Identification of intramembrane hydrogen bonding between 13(1) keto group of bacteriochlorophyll and serine residue alpha27 in the LH2 light-harvesting complex.

Intramembrane hydrogen bonding and its effect on the structural integrity of purple bacterial light-harvesting complex 2, LH2, have been assessed in the native membrane environment. A novel hydrogen bond has been identified by Raman resonance spectroscopy between a serine residue of the membrane-spanning region of LH2 alpha-subunit, and the C-13(1) keto carbonyl of bacteriochlorophyll (BChl) B850 bound to the beta-subunit. Replacement of the serine by alanine disrupts this strong hydrogen bond, but this neither alters the strongly red-shifted absorption nor the structural arrangement of the BChls, as judged from circular dichroism. It also decreases only slightly the thermal stability of the mutated LH2 in the native membrane environment. The possibility is discussed that weak H-bonding between the C-13(1) keto carbonyl and a methyl hydrogen of the alanine replacing serine(-4) or the imidazole group of the nearby histidine maintains structural integrity in this very stable bacterial light-harvesting complex. A more widespread occurrence of H-bonding to C-13(1) not only in BChl, but also in chlorophyll proteins, is indicated by a theoretical analysis of chlorophyll/polypeptide contacts at <3.5 A in the high-resolution structure of Photosystem I. Nearly half of the 96 chlorophylls have aa residues suitable as hydrogen bond donors to their keto groups.

[High resolution EEG and whole head MEG]. / Hochauflösendes EEG im Ganzkopf-MEG.

A low-noise multichannel EEG-Amplifier-System has been designed and analyzed, which is electromagnetically compatible with SQUIDs. The EEG- together with the MEG-System is operated inside the quiet environment of a magnetically shielded room, hence no electrical 50 Hz-Artifact is detectable. Measurements pointed out that the influence of amplifier- and electrochemical-noise of electrodes to averaged EEG-spectral densities is less than 1% within the frequency range 0.5 to 70 Hz. Thermal noise of the skin-electrode-interface, equivalent to a resistor of 30 k omega, only begins to take effect above 20 Hz, suggesting that there is no need of skin abrasion.

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically 'dark') S(1) state of carotenoids.

Native and carotenoid-depleted peripheral purple bacterial light-harvesting complex (LH2) were investigated by simultaneous two-photon excited (between 1300-1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2A(g)(-) state of rhodopin [corrected] is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid-depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.

Assembly of light-harvesting bacteriochlorophyll in a model transmembrane helix in its natural environment.

The transmembrane, bacteriochlorophyll-binding region of a bacterial light-harvesting complex, (LH2-alpha from the photosynthetic bacterium Rhodobacter sphaeroides) was redesigned and overexpressed in a mutant of Rb. sphaeroides lacking LH2. Bacteriochlorophyll served as internal probe for the fitness of this new region for the assembly and energy transfer function of the LH2 complex. The ability to absorb and transfer light energy is practically undisturbed by the exchange of the transmembrane segment, valine -7 to threonine +6, of LH2-alpha with a 14 residue Ala-Leu sequence. This stretch makes up the residues of the transmembrane helix that are in close contact (< or =4.5 A) with the bacteriochlorophyll molecules that are coordinated through His of both the alpha and beta-subunits. In this Ala-Leu stretch, neither alpha-His0, which binds the bacteriochlorophyll, nor the adjacent alpha-Ile-1, were replaced. Novel LH2 complexes composed of LH2-alpha with a model transmembrane sequence and a normal LH2-beta are assembled in vivo into a complex, the biochemical and spectroscopic properties of which closely resemble the native one. In contrast, the additional insertion of four residues just outside the C-terminal end of the model transmembrane helix leads to complete loss of functional antenna complex. The results suggest that light energy can be harvested and transferred efficiently by bacteriochlorophyll molecules attached to only few key residues distributed over the polypeptide, while residues at the bacteriochlorophyll-helix interface seem to be largely dispensable for the functional assembly of this membrane protein complex. This novel antenna with a simplified transmembrane domain and a built-in probe for assembly and function provides a powerful model system for investigation of the factors that contribute to the assembly of chromophores in membrane-embedded proteins.

Spectroscopy of single phycoerythrocyanin monomers: dark state identification and observation of energy transfer heterogeneities.

Phycoerythrocyanin (PEC) is part of the light harvesting system of cyanobacteria. The PEC monomer contains one phycoviolobilin chromophore, which transfers excitation energy onto two phycocyanobilin chromophores. Many spectroscopical methods have been used in the past to study the bulk properties of PEC. These methods average over many molecules. Therefore, differences in the behavior of individual molecules remain hidden. The energy transfer within photosynthetic complexes is however sensitive to changes in the spectroscopic properties of the participating subunits. Knowledge about heterogeneities is therefore important for the description of the energy transfer in photosynthetic systems. Here, the recording of the fluorescence emission of single PEC molecules is used as a tool to obtain such information. Spectrally resolved detection as well as double resonance excitation of single PEC molecules is used to investigate their bleaching behavior. The trans isomer of the phycoviolobilin chromophore is identified as a short-lived dark state of monomeric PEC. Polarization sensitive single molecule detection is used for the direct observation of the energy transfer in individual PEC molecules. The experiments reveal that more than one-half of the PEC molecules exhibit an energy transfer behavior significantly different from the bulk. These heterogeneities persist on a time scale of several seconds. Model calculations lead to the conclusion that they are caused by minor shifts in the spectra of the chromophores.