Fluorescence spectroscopy of conformational changes of single LH2 complexes

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between long-lived quasi-stable levels differing by up to 30 nm. The frequency and size of these fluorescence peak movements were found to increase linearly with the excitation intensity. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The probability for a particle to undergo significant spectral shift in either direction was found to be roughly the same. Using the modified Redfield theory, the observed changes in spectral shape and intensity were accounted for by changes in the realization of the static disorder. Long lifetimes of the quasi-stable states suggest large energetic barriers between the states characterized by different emission spectra.     

Fluorescence spectral fluctuations of single LH2 complexes from Rhodopseudomonas acidophila strain 10050

We have investigated the energy landscape of the bacterial photosynthetic peripheral light-harvesting complex LH2 of purple bacterium Rhodopseudomonas acidophila by monitoring sequences of fluorescence spectra of single LH2 assemblies, at room temperature, with different excitation intensities as well as at elevated temperatures, utilizing a confocal microscope. The fluorescence peak wavelength of individual LH2 complexes was found to abruptly move between quasi-stable levels differing by up to 30 nm. These spectral shifts either to the blue or to the red were accompanied by a broadening and decrease of the intensity of the fluorescence spectrum. The frequency and size of these fluorescence peak movements were found to increase linearly with excitation intensity. Using the modified Redfield theory, changes in the realization of the static disorder accounted for the observed changes in spectral shape and intensity. Long lifetimes of the quasi-stable states suggest large free energy barriers between the different realizations.     

Spectral trends in the fluorescence of single bacterial light-harvesting complexes: experiments and modified redfield simulations

In this work we present and discuss the single-molecule fluorescence spectra of a variety of species of light-harvesting complexes: LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum and LH1 of Rhodobacter sphaeroides. The emission spectrum of these complexes varies as a function of time as was described in earlier work. For each type of complex, we observe a pronounced and well-reproducible characteristic relationship between the fluorescence spectral parameters of the peak wavelength, width, and asymmetry. This dependence for the LH2 complexes can be quantitatively explained on the basis of a disordered exciton model by varying the static disorder and phonon coupling parameters. In addition, a correlation of the pigment site energies has to be assumed to interpret the behavior of the LH1 complex.     

Dynamics of the emission spectrum of a single LH2 complex: interplay of slow and fast nuclear motions

We have studied the relationship between the realizations of static disorder and the emission spectra observed for a single LH2 complex. We show that the experimentally observed spectral fluctuations reflect realizations of the disorder in the B850 ring associated with different degrees of exciton delocalization and different effective coupling of the excitons to phonon modes. The main spectral features cannot be explained using models with correlated disorder associated with elliptical deformations of the ring. A quantitative explanation of the measured single-molecule spectra is obtained using the modified Redfield theory and a model of the B850 ring with uncorrelated disorder of the site energies. The positions and spectral shapes of the main exciton components in this model are determined by the disorder-induced shift of exciton eigenvalues in combination with phonon-induced effects (i.e., reorganization shift and broadening, that increase in proportion to the inverse delocalization length of the exciton state). Being dependent on the realization of the disorder, these factors produce different forms of the emission profile. In addition, the different degree of delocalization and effective couplings to phonons determines a different type of excitation dynamics for each of these realizations. We demonstrate that experimentally observed quasistable conformational states are characterized by excitation energy transfer regimes varying from a coherent wavelike motion of a delocalized exciton (with a 100-fs pass over half of the ring) to a hopping-type motion of the wavepacket (with a 350-fs jump between separated groups of 3-4 molecules) and self-trapped excitations that do not move from their localization site.     

Probing the stability of the SpCas9–DNA complex after cleavage

CRISPR–Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.     

Energy transfer in the peridinin chlorophyll-a protein of Amphidinium carterae studied by polarized transient absorption and target analysis

The peridinin chlorophyll-a protein (PCP) of dinoflagellates differs from the well-studied light-harvesting complexes of purple bacteria and green plants in its large (4:1) carotenoid to chlorophyll ratio and the unusual properties of its primary pigment, the carotenoid peridinin. We utilized ultrafast polarized transient absorption spectroscopy to examine the flow of energy in PCP after initial excitation into the strongly allowed peridinin S2 state. Global and target analysis of the isotropic and anisotropic decays reveals that significant excitation (25-50%) is transferred to chlorophyll-a directly from the peridinin S2 state. Because of overlapping positive and negative features, this pathway was unseen in earlier single-wavelength experiments. In addition, the anisotropy remains constant and high in the peridinin population, indicating that energy transfer from peridinin to peridinin represents a minor or negligible pathway. The carotenoids are also coupled directly to chlorophyll-a via a low-lying singlet state S1 or the recently identified SCT. We model this energy transfer time scale as 2.3 +/- 0.2 ps, driven by a coupling of approximately 47 cm(-1). This coupling strength allows us to estimate that the peridinin S1/SCT donor state transition moment is approximately 3 D.     

Conformational relaxation of single bacterial light-harvesting complexes

We have employed the technique of single-molecule fluorescence microspectroscopy to investigate the spontaneous conformational evolution of individual peripheral LH2 complexes from the purple bacterium Rhodopseudomonas acidophila. Fluorescence microscopy is a sensitive tool, which allows the spectral changes of single complexes to be monitored on a time scale from 0.1 s to many minutes. Here we have investigated "natural" (occurring in the absence of excitation) spectral diffusion after a spectral jump has occurred. In a quarter of all the observed spectral jumps recorded with the LH2 complexes, a further spontaneous evolution occurs, in the absence of illumination, that results in the formation of a different spectroscopic state. We suggest that this is due to a natural conformational development of the pigment-protein complex, which so far has not been observed for this type of complex at the single-molecule level. The functional significance of such structural rearrangements is not yet clear but may be associated with the necessity for the light-harvesting complexes to adjust their shape in the densely packed photosynthetic membrane.     

Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI

Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5′-W/CCGGW-3′ (W stands for A or T, ‘/’ denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an ‘open’ configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes.     

Comparative study of spectral flexibilities of bacterial light-harvesting complexes: structural implications

This work presents a comparative study of the frequencies of spectral jumping of individual light-harvesting complexes of six different types: LH2 of Rhodopseudomonas acidophila, Rhodobacter sphaeroides, and Rhodospirillum molischianum; LH1 of Rhodobacter sphaeroides; and two "domain swap mutants" of LH2 of Rhodobacter sphaeroides: PACLH1 and PACLH2mol, in which the alpha-polypeptide C-terminus is exchanged with the corresponding sequence from LH1 of Rhodobacter sphaeroides or LH2 of Rhodospirillum molischianum, respectively. The quasistable states of fluorescence peak wavelength that were previously observed for the LH2 of Rps. acidophila were confirmed for other species. We also observed occurrences of extremely blue-shifted spectra, which were associated with reversible bleaching of one of the chromophore rings. Different jumping behavior is observed for single complexes of different types investigated with the same equivalent excitation intensity. The differences in spectral diffusion are associated with subtle differences of the binding pocket of B850 pigments and the structural flexibility of the different types of complexes.     

Spectral dynamics of individual bacterial light-harvesting complexes: alternative disorder model

The bacterial (Rhodopseudomonas acidophila) photosynthetic peripheral light-harvesting complex of type 2 (LH2) exhibits rich fluorescence spectral dynamics at room temperature. The fluorescence spectrum of individual LH2 shifts either to the blue or to the red during the experimental observation time of a few minutes. These spectral changes are often reversible and occur between levels of a distinctly different peak wavelength. Furthermore, they are accompanied by a change of the spectral line shape. To interpret the dynamics of spectral changes, an energetic disorder model associated with easily explainable structural changes of the protein is proposed. This model assumes that each pigment in the tightly coupled ring of bacteriochlorophylls can be in two states of electronic transition energy due to the protein-pigment interaction. The transition between these structural, and hence spectroscopic, states occurs through the thermally induced conformational potential energy barrier crossing. Although simplified, the model allows us to reproduce the bulk fluorescence spectrum, the distribution of the single-molecule spectral peak wavelength and its changes, and the statistics of the duration of the spectral states. It also provides an intuitively clear picture of possible protein dynamics in LH2. At the same time, it requires additional sophistication since it essentially does not reproduce the red occurrences of single LH2 spectra.