Tjaart P.J. Krüger

Fluorescence Spectral Dynamics of Single LHCII Trimers

Single-molecule spectroscopy was employed to elucidate the fluorescence spectral heterogeneity and dynamics of individual, immobilized trimeric complexes of the main light-harvesting complex of plants in solution near room temperature. Rapid reversible spectral shifts between various emitting states, each of which was quasi-stable for seconds to tens of seconds, were observed for a fraction of the complexes. Most deviating states were characterized by the appearance of an additional, red-shifted emission band. Reversible shifts of up to 75 nm were detected. By combining modified Redfield theory with a disordered exciton model, fluorescence spectra with peaks between 670 nm and 705 nm could be explained by changes in the realization of the static disorder of the pigment-site energies. Spectral bands beyond this wavelength window suggest the presence of special protein conformations. We attribute the large red shifts to the mixing of an excitonic state with a charge-transfer state in two or more strongly coupled chlorophylls. Spectral bluing is explained by the formation of an energy trap before excitation energy equilibration is completed.

A Fisk-Parker Hybrid Heliospheric Magnetic Field with a Solar-Cycle Dependence

We present a refinement of the Fisk-Parker hybrid field of Burger and Hitge which now includes a region bordering the solar rotational equator where magnetic field footpoint motion occurs only through diffusive reconnection. The hybrid field, therefore, only occurs above a certain latitude in a given hemisphere, and in the equatorial region the field is a pure Parker field. We also propose a simple qualitative model for the solar cycle dependence of the hybrid field, taking into account changes in the tilt angle of the heliospheric current sheet and the latitudinal extend of the polar coronal hole on the photosphere and on the source surface over the course of a solar activity cycle. We find that the amplitude of magnetic field fluctuations for assumed solar minimum parameters would not be observable above the background noise (see Roberts and coworkers). We also show that for these parameters, periodicities associated with differential footpoint motion would be barely distinguishable from rigid rotation at the solar equatorial rate. We point out that the question of periodicities in magnetic field data is perhaps more complicated than previously thought. We confirm the result of Burger and Hitge that a Fisk-type heliospheric magnetic field provides a natural explanation for the observed linear relationship between the amplitude of the recurrent cosmic-ray variations and the global latitude gradient (see Zhang). We show that this relationship holds for helium, protons, and electrons. Moreover, we show that the constant of proportionality is larger when qA > 0 than when qA < 0, as inferred from observations by Richardson and coworkers.

Controlled Disorder in Plant Light-Harvesting Complex II Explains Its Photoprotective Role

The light-harvesting antenna of photosystem II (PSII) has the ability to switch rapidly between a state of efficient light use and one in which excess excitation energy is harmlessly dissipated as heat, a process known as qE. We investigated the single-molecule fluorescence intermittency of the main component of the PSII antenna (LHCII) under conditions that mimic efficient use of light or qE, and we demonstrate that weakly fluorescing states are stabilized under qE conditions. Thus, we propose that qE is explained by biological control over the intrinsic dynamic disorder in the complex-the frequencies of switching establish whether the population of complexes is unquenched or quenched. Furthermore, the quenched states were accompanied by two distinct spectral signatures, suggesting more than one mechanism for energy dissipation in LHCII.

Conformational switching explains the intrinsic multifunctionality of plant light-harvesting complexes

The light-harvesting complexes of photosystem I and II (Lhcas and Lhcbs) of plants display a high structural homology and similar pigment content and organization. Yet, the spectroscopic properties of these complexes, and accordingly their functionality, differ substantially. This difference is primarily due to the charge-transfer (CT) character of a chlorophyll dimer in all Lhcas, which mixes with the excitonic states of these complexes, whereas this CT character is generally absent in Lhcbs. By means of single-molecule spectroscopy near room temperature, we demonstrate that the presence or absence of such a CT state in Lhcas and Lhcbs can occasionally be reversed; i.e., these complexes are able to interconvert conformationally to quasi-stable spectral states that resemble the Lhcs of the other photosystem. The high structural similarity of all the Lhca and Lhcb proteins suggests that the stable conformational states that give rise to the mixed CT-excitonic state are similar for all these proteins, and similarly for the conformations that involve no CT state. This indicates that the specific functions related to Lhca and Lhcb complexes are realized by different stable conformations of a single generic protein structure. We propose that this functionality is modulated and controlled by the protein environment.

Fluorescence Intermittency from the Main Plant Light-Harvesting Complex: Sensitivity to the Local Environment

The time-resolved fluorescence intensity fluctuations from single, immobilized complexes of the main light-harvesting complex (LHCII) of plants were investigated in different pH environments close to room temperature and under different light conditions. The efficiency of light harvesting, which was represented by complexes typically residing for long periods in strongly fluorescing states, was significantly reduced by decreasing the pH or increasing the incident photon flux. The same environmental changes significantly increased the switching frequency between strongly and weakly fluorescing states. The environmental dependence became more evident when the various accessed intensity levels were first resolved, a technique that significantly reduced the obscuring effect of shot noise. The strong environmental sensitivity suggests that the immediate environment of an LHCII complex can modulate the amount of energy dissipation. A simple model illustrates how this may be achieved: the dynamic equilibrium between the strongly and weakly fluorescing states can be shifted by environmentally controlling the conformational diffusion on the potential energy surface of LHCII.

Disentangling the low-energy states of the major light-harvesting complex of plants and their role in photoprotection☆

The ability to dissipate large fractions of their absorbed light energy as heat is a vital photoprotective function of the peripheral light-harvesting pigment-protein complexes in photosystem II of plants. The major component of this process, known as qE, is characterised by the appearance of low-energy (red-shifted) absorption and fluorescence bands. Although the appearance of these red states has been established, the molecular mechanism, their site and particularly their involvement in qE are strongly debated. Here, room-temperature single-molecule fluorescence spectroscopy was used to study the red emission states of the major plant light-harvesting complex (LHCII) in different environments, in particular conditions mimicking qE. It was found that most states correspond to peak emission at around 700nm and are unrelated to energy dissipative states, though their frequency of occurrence increased under conditions that mimicked qE. Longer-wavelength emission appeared to be directly related to energy dissipative states, in particular emission beyond 770nm. The ensemble average of the red emission bands shares many properties with those obtained from previous bulk in vitro and in vivo studies. We propose the existence of at least three excitation energy dissipating mechanisms in LHCII, each of which is associated with a different spectral signature and whose contribution to qE is determined by environmental control of protein conformational disorder. Emission at 700nm is attributed to a conformational change in the Lut 2 domain, which is facilitated by the conformational change associated with the primary quenching mechanism involving Lut 1.

Single-Molecule Identification of Quenched and Unquenched States of LHCII.

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.

Fluorescence intermittency from the main plant light-harvesting complex: resolving shifts between intensity levels.

We present a simple method to resolve discrete intensity shifts from time-resolved single-molecule emission data. This new method uses multiples of the standard deviation of the measured intensities that are integrated into short time bins. By applying the technique to trimeric units of the main light-harvesting complex (LHCII) of plants, it is shown that the amount of information that can be extracted from an intensity time trace increases considerably, thereby enlarging the possibility to reveal new phenomena. It is demonstrated how shot noise can lead to substantial deviations and misleading interpretations when the conventional two-state kinetic model for intensity fluctuations is applied. By first resolving the accessed intensity levels, the artifactual effect of shot noise is sufficiently reduced. The technique is particularly applicable to the analysis of fluorescence intermittency from multichromophoric systems.

Controlling Light Harvesting with Light

When exposed to intense sunlight, all organisms performing oxygenic photosynthesis implement various photoprotective strategies to prevent potentially lethal photodamage. The rapidly responding photoprotective mechanisms, occurring in the light-harvesting pigment-protein antennae, take effect within tens of seconds, while the dramatic and potentially harmful light intensity fluctuations manifest also on shorter time scales. Here we show that, upon illumination, individual phycobilisomes from Synechocystis PCC 6803, which, in vivo under low-light conditions, harvest solar energy, and have the built-in capacity to switch rapidly and reversibly into light-activated energy-dissipating states. Simultaneously measured fluorescence intensity, lifetime, and spectra, compared with a multicompartmental kinetic model, revealed that essentially any subunit of a phycobilisome can be quenched, and that the core complexes were targeted most frequently. Our results provide the first evidence for fluorescence blinking from a biologically active system at physiological light intensities and suggest that the light-controlled switches to intrinsically available energy-dissipating states are responsible for a novel type of photoprotection in cyanobacteria. We anticipate other photosynthetic organisms to employ similar strategies to respond instantly to rapid solar light intensity fluctuations. A detailed understanding of the photophysics of photosynthetic antenna complexes is of great interest for bioinspired solar energy technologies.

Mid-ATR-FTIR spectroscopic profiling of HIV/AIDS sera for novel systems diagnostics in global health.

Global health, whether in developed or developing countries, is in need of robust systems diagnostics for major diseases, such as HIV/AIDS, impacting the world populations. Fourier transform Infrared (FTIR) spectroscopy of serum is a quick and reagent-free methodology with which to analyze metabolic alterations such as those caused by disease or treatment. In this study, Attenuated Total Reflectance Fourier-Transform (ATR-FTIR) Spectroscopy was investigated as a means of distinguishing HIV-infected treatment-experienced (HIV(pos) ART(pos), n=39) and HIV-infected-treatment-naïve (HIV(pos) ART(neg), n=16) subjects from uninfected control subjects (n=30). Multivariate pattern recognition techniques, including partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares discriminant analysis (OPLS-DA), successfully distinguished sample classes, while univariate approaches identified significant differences (p<0.05) after Benjamini-Hochberg corrections. OPLS-DA discriminated between all groups with sensitivity, specificity, and accuracy of >90%. Compared to uninfected controls, HIV(pos) ART(pos) and HIV(pos) ART(neg) subjects displayed significant differences in spectral regions linked to lipids/fatty acids (3010 cm(-1)), carbohydrates (1299 cm(-1); 1498 cm(-1)), glucose (1035 cm(-1)), and proteins (1600 cm(-1); 1652 cm(-1)). These are all molecules shown by conventional biochemical analysis to be affected by HIV/ART interference. The biofluid metabolomics approach applied here successfully differentiated global metabolic profiles of HIV-infected patients and uninfected controls and detected potential biomarkers for development into indicators of host response to treatment and/or disease progression. Our findings therefore contribute to ongoing efforts for capacity-building in global health for robust omics science and systems diagnostics towards major diseases impacting population health.